Multiple-tumor aberrant growth genes

ABSTRACT

The invention relates to the multi-tumor aberrant growth gene having the nucleotide sequence of any one of the strands of any one of the members of the High Mobility Group protein genes or LIM protein genes, including modified versions and derivatives thereof. The gene and its derivatives may be used in various diagnostic and therapeutic applications.

[0001] The present invention relates to the identification of the HighMobility Group (HMG) protein gene family as a family of genes frequentlyassociated with aberrant cell growth as found in a variety of bothbenign and malignant tumors. The invention in particular relates to theidentification of a member of the HMG gene family as the broadly actingchromosome 12 breakpoint region gene involved in a number of tumors,including but not limited to the mesenchymal tumors hamartomas (e.g.breast and lung), lipomas, pleomorphic salivary gland adenomas, uterineleiomyomas, angiomyxomas, fibroadenomas of the breast, polyps of theendometrium, atherosclerotic plaques, and other benign tumors as well asvarious malignant tumors, including but not limited to sarcomas (e.g.rhabdomyosarcoma, osteosarcoma) and carcinomas (e.g. of breast, lung,skin, thyroid), as well as leukemias and lymphomas. The invention alsorelates to another member of the HMG gene family that was found to beimplicated in breaks in chromosome 6.

[0002] Furthermore, the invention concerns the identification of membersof the LIM protein family as another type of tumor-type specificbreakpoint region genes and frequent fusion partners of the HMG genes inthese tumors. The LPP (Lipoma-Preferred Partner) gene of this family isfound to be specific for lipomas. The invention relates in particular tothe use of the members of the HMG and LIM gene family and theirderivatives in diagnosis and therapy.

[0003] Multiple independent cytogenetic studies have firmly implicatedregion q13-q15 of chromosome 12 in a variety of benign and malignantsolid tumor types. Among benign solid tumors, involvement of 12q13-q15is frequently observed in benign adipose tissue tumors [1], uterineleiomyomas [2, 3], and pleomorphic adenomas of the salivary glands [4,5]. Involvement of the same region has also been reported forendometrial polyps [6, 7] for hemangiopericytoma [8], and forchondromatous tumors [9, 10, 11, 12]. Recently, the involvement ofchromosome 12q13-q15 was reported in pulmonary chondroid hamartoma [13,14]. Finally, several case reports of solid tumors with involvement ofchromosome region 12q13-q15 have been published; e.g. tumors of thebreast [15, 16], diffuse astrocytomas [17], and a giant-cell tumor ofthe bone [18]. Malignant tumor types with recurrent aberrations in12q13-q15 include myxoid liposarcoma [19], soft tissue clear-cellsarcoma [20, 21, 22], and a subgroup of rhabdomyosarcoma [23].

[0004] Although these studies indicated that the same cytogenetic regionof chromosome 12 is often involved in chromosome aberrations, liketranslocations, in these solid tumors, the precise nature of thechromosome 12 breakpoints in the various tumors is still not known.Neither was it established which genes are affected directly by thetranslocations.

[0005] In previous physical mapping studies [39], the chromosome 12qbreakpoints in lipoma, pleomorphic salivary gland adenoma, and uterineleiomyoma were mapped between locus D12S8 and the CHOP gene and it wasshown that D12S8 is located distal to CHOP. Recently, it was also foundby FISH analysis that the chromosome 12q breakpoints in a hamartoma ofthe breast, an angiomyxoma and multiple pulmonary chondroid hamartomasare mapping within this DNA interval. In an effort to molecularly clonethe genes affected by the chromosome 12q13-q15 aberrations in thevarious tumors, the present inventors chose directional chromosomewalking as a structural approach to define the DNA region encompassingthese breakpoints.

[0006] As a starting point for chromosome walking, locus D12S8 was used.During these walking studies, it was shown that the chromosomalbreakpoints as present in a number of uterine leiomyoma-derived celllines are clustered within a 445 kb chromosomal segment which has beendesignated Uterine Leiomyoma Cluster Region on chromosome 12 (ULCR12)[24]. Subsequently, it was found that a 1.7 Mb region on chromosome 12encompasses the chromosome 12 breakpoints of uterine leiomyoma-,lipoma-, and salivary gland adenoma-cells, with the breakpoint clusterregions of the various tumor types overlapping [25, “ANNEX 1”]. This 1.7Mb region on the long arm of chromosome 12, which contains ULCR12obviously, was designated Multiple Aberration Region (MAR) to reflectthis feature. In a regional fine mapping study, MAR was recentlyassigned to 12q15.

[0007] It has thus been found that essentially all breakpoints ofchromosome 12 map in a 1.7 Mb region indicated herein as the “MultipleAberration Region” or MAR. Further research revealed that in this regiona member of the High Mobility Group gene family, the HMGI-C gene, can beidentified as a postulated multi-tumor aberrant growth gene (MAG). Thesame applies to members of the LIM family which are also found to beinvolved in chromosome aberrations. Of these the chromosome 3-derivedLipoma-Preferred Partner (LPP) gene is particularly implicated inlipomas.

[0008] LIM proteins are proteins carrying cystein-rich zinc-bindingdomains, so-called LIM domains. They are involved in protein-proteininteractions [for a review see ref. 80]. One of the members of theprotein family is the now disclosed LPP protein mapping at chromosome 3.

[0009] According to the present invention the aberrations in the HMGI-Cgene on chromosome 12 and the LPP gene on chromosome 3 have been used asa model to reveal the more general concept of the involvement of membersof the HMG and LIM gene families in both benign and malignant tumors. Todemonstrate that there exists a more general concept of gene families,which, when affected by chromosome rearrangements, lead to a particulargroup of tumor growth, the present inventors demonstrated that theHMGI(Y) gene, which is a member of the HMG family, is involved in breaksin chromosome 6.

[0010] Although both the HMG and LIM gene families are known per se, uptill the present invention the correlation between these families andtumor inducing chromosome aberrations, like translocations, deletions,insertions and inversions, has not been anticipated. Furthermore, untilnow it was not previously demonstrated that alterations in thephysiological expression level of the members of the gene family areprobably also implicated in tumor development. According to theinvention it was demonstrated that in normal adult cells the expressionlevel of the HMGI-C gene is practically undetectable, whereas inaberrantly growing cells the expression level is significantlyincreased.

[0011] Based on these insights the present invention now provides forthe members of the gene families or derivatives thereof in isolated formand their use in diagnostic and therapeutic applications. Furthermorethe knowledge on the location and nucleotide sequence of the genes maybe used to study their rearrangements or expression and to identify apossible increase or decrease in their expression level and the effectsthereof on cell growth. Based on this information diagnostic tests ortherapeutic treatments may be designed.

[0012] In this application the term “Multi-tumor Aberrant Growth (orMAG) gene” will be used to indicate the involvement of these types ofgenes in various types of cancer. The term refers to all members of theHMG and LIM gene families involved in non-physiological proliferativegrowth, and in particular involved in malignant or benign tumors,including atherosclerotic plaques. However, according to the inventionit was furthermore found that even breaks outside the actual gene but inthe vicinity thereof might result in aberrant growth. The term MAG geneis therefore also intended to include the immediate vicinity of thegene. The skilled person will understand that the “immediate vicinity”should be understood to include the surroundings of the gene in whichbreaks or rearrangements will result in the above definednon-physiological proliferative growth.

[0013] The term “wildtype cell” is used to indicate the cell notharbouring an aberrant chromosome or to a cell having a physiologicalexpression level of the relevant gene. “Wildtype” or “normal” chromosomerefers to a non-aberrant chromosome.

[0014] The present invention provides for various diagnostic andtherapeutic applications that are based on the information that may bederived from the genes. This information not only encompasses itsnucleotide sequence or the amino acid sequence of the gene productderived from the gene, but also involves the levels of transcription ortranslation of the gene.

[0015] The invention is thus two-fold. On the one hand the aberration incell growth may be directly or indirectly caused by the physical breaksthat occur in the gene or its vicinity. On the other hand the aberrationin cell growth may be caused by a non-physiological expression level ofthe gene. This non-physiological expression level may be caused by thebreak, or may be due to another stimulus that activates or deactivatesthe gene. At present the exact mechanism or origin of the aberrant cellgrowth is not yet unraveled. However, exact knowledge on this mechanismis not necessary to define methods of diagnosis or treatment.

[0016] Diagnostic methods according to the invention are thus based onthe fact that an aberration in a chromosome results in a detectablealteration in the chromosomes' appearance or biochemical behaviour. Atranslocation, for example will result in a first part of the chromosome(and consequently of the MAG gene) having been substituted for another(second) part (further referred to as “first and second substitutionparts”). The first part will often appear someplace else on anotherchromosome from which the second part originates. As a consequencehybrids will be formed between the remaining parts of both (or in casesof triple translocations, even more) chromosomes and the substitutionparts provided by their translocation partners. Since it has now beenfound that the breaks occur in a MAG gene this will result in hybridgene products of that MAG gene. Markers, such as hybridising moleculeslike RNA, DNA or DNA/RNA hybrids, or antibodies will be able to detectsuch hybrids, both on the DNA level, and on the RNA or protein level.

[0017] For example, the transcript of a hybrid will still comprise theregion provided by the remaining part of the gene/chromosome but willmiss the region provided by the substitution part that has beentranslocated. In the case of inversions, deletions and insertions thegene may be equally afflicted.

[0018] Translocations are usually also cytogenetically detectable. Theother aberrations are more difficult to find because they are often notvisible on a cytogenetical level. The invention now providespossibilities for diagnosing all these types of chromosomal aberrations.

[0019] In translocations markers or probes based on the MAG gene for theremaining and substitution parts of a chromosome in situ detect theremaining part on the original chromosome but the substitution part onanother, the translocation partner.

[0020] In the case of inversions for example, two probes will hybridiseat a specific distance in the wildtype gene. This distance might howeverchange due to an inversion. In situ such inversion may thus bevisualized by labeling a set of suitable probes with the same ordifferent detectable markers, such as fluorescent labels. Deletions andinsertions may be detected in a similar manner.

[0021] According to the invention the above in situ applications canvery advantageously be performed by using FISH techniques. The markersare e.g. two cosmids one of which comprises exons 1 to 3 of the MAGgene, while the other comprises exons 4 and 5. Both cosmids are labeledwith different fluorescent markers, e.g. blue and yellow. The normalchromosome will show a combination of both labels, thus giving a greensignal, while the translocation is visible as a blue signal on theremaining part of one chromosome (e.g. 12) while the yellow signal isfound on another chromosome comprising the substitution part. In casethe same labels are used for both probes, the intensity of the signal onthe normal chromosome will be 100%, while the signal on the aberrantchromosomes is 50%. In the case of inversions one of the signals shiftsfrom one place on the normal chromosome to another on the aberrant one.

[0022] In the above applications a reference must be included forcomparison. Usually only one of the two chromosomes is afflicted. Itwill thus be very convenient to use the normal chromosome as an internalreference. Furthermore it is important to select one of the markers onthe remaining or unchanging part of the chromosome and the other on thesubstitution or inverted part. In the case of the MAG gene of chromosome12, breaks are usually found in the big intron between exons 3 and 4 asis shown by the present invention. Furthermore breaks were detectedbetween exons 4 and 5. Probes based on exons 1 to 3 and 4 and 5, orprobes based on either exon 4 or on exon 5 are thus very useful. As analternative a combination of probes based on both translocation orfusion partners may be used. For example, for the identification oflipomas one may use probes based on exons 1 to 3 of the HMGI-C gene onthe one hand and based on the LIM domains of the LPP gene on the otherhand.

[0023] Furthermore it was found that breaks might also occurr outsidethe gene, i.e. 5′ or 3′ thereof. The choice of the probes will then ofcourse include at least one probe hybrising to a DNA sequence located 5′or 3′ of the gene.

[0024] “Probes” as used herein should be widely interpreted and includebut are not limited to linear DNA or RNA strands, Yeast ArtificialChromosomes (YACs), or circular DNA forms, such as plasmids, phages,cosmids etc.

[0025] These in situ methods may be used on metaphase and interphasechromosomes.

[0026] Besides the above described in situ methods various diagnostictechniques may be performed on a more biochemical level, for examplebased on alterations in the DNA, RNA or protein, or on changes in thephysiological expression level of the gene.

[0027] Basis for the methods that are based on alterations in thechromosome's biochemical behaviour is the fact that by choosing suitableprobes, variations in the length or composition in the gene, transcriptor protein may be detected on a gel or blot. Variations in length arevisible because the normal gene, transcript(s) or protein(s) will appearin another place on the gel or blot then the aberrant one(s). In case ofa translocation more than the normal number of spots will appear.

[0028] Based on the above principle the invention may thus for examplerelate to a method of diagnosing cells having a non-physiologicalproliferative capacity, comprising the steps of taking a biopsy of thecells to be diagnosed, isolating a suitable MAG gene-relatedmacromolecule therefrom, and analysing the macromolecule thus obtainedby comparison with a reference molecule originating from cells notshowing a non-physiological proliferative capacity, preferably from thesame individual. The MAG gene-related macromolecule may thus be a DNA,an RNA or a protein. The MAG gene may be either a member of the HMGfamily or of the LIM family.

[0029] In a specific embodiment of this type of diagnostic method theinvention comprises the steps of taking a biopsy of the cells to bediagnosed, extracting total RNA thereof, preparing a first strand cDNAof the mRNA species in the total RNA extract or poly-A-selectedfraction(s) thereof, which cDNA comprises a suitable tail; performing aPCR using a MAG gene specific primer and a tail-specific primer in orderto amplify MAG gene specific cDNA's; separating the PCR products on agel to obtain a pattern of bands; evaluating the presence of aberrantbands by comparison to wildtype bands, preferably originating from thesame individual.

[0030] As an alternative amplification may be performed by means of theNucleic Acid Sequence-Based Amplification (NASBA) technique [81] orvariations thereof.

[0031] In another embodiment the method comprises the steps of taking abiopsy of the cells to be diagnosed, isolating total protein therefrom,separating the total protein on a gel to obtain essentially individualbands, optionally transfering the bands to a Western blot, hybridisingthe bands thus obtained with antibodies directed against a part of theprotein encoded by the remaining part of the MAG gene and against a partof the protein encoded by the substitution part of the MAG gene;visualising the antigen-antibody reactions and establishing the presenceof aberrant bands by comparison with bands from wildtype proteins,preferably originating from the same individual.

[0032] In a further embodiment the method comprises taking a biopsy ofthe cells to be diagnosed; isolating total DNA therefrom; digesting theDNA with one or more so-called “rare cutter” restriction enzymes(typically “6- or more cutters”); separating the digest thus prepared ona gel to obtain a separation pattern; optionally transfering theseparation pattern to a Southern blot; hybridising the separationpattern in the gel or on the blot with a set of probes under hybridisingconditions; visualising the hybridisations and establishing the presenceof aberrant bands by comparison to wildtype bands, preferablyoriginating from the same individual.

[0033] Changes in the expression level of the gene may be detected bymeasuring mRNA levels or protein levels by means of a suitable probe.

[0034] Diagnostic methods based on abnormal expression levels of thegene may comprise the steps of taking a sample of the cells to bediagnosed; isolating mRNA therefrom; and establishing the presenceand/or the (relative) quantity of mRNA transcribed from the MAG gene ofinterest in comparison to a control. Establishing the presence or(relative) quantity of the mRNA may be achieved by amplifying at leastpart of the mRNA of the MAG gene by means of RT-PCR or similaramplification techniques. In an alternative embodiment the expressionlevel may be established by determination of the presence or the amountof the gene product (e.g. protein) by means of for example monoclonalantibodies.

[0035] The diagnostic methods of the invention may be used for diseaseswherein cells having a non-physiological proliferative capacity areselected from the group consisting of benign tumors, such as themesenchymal tumors hamartomas (e.g. breast and lung), adipose tissuetumors (e.g. lipomas), pleomorphic salivary gland adenomas, uterineleiomyomas, angiomyxomas, fibroadenomas of the breast, polyps of theendometrium, atherosclerotic plaques, and other benign tumors as well asvarious malignant tumors, including but not limited to sarcomas (e.g.rhabdomyosarcoma, osteosarcoma) and carcinomas (e.g. of breast, lung,skin, thyroid). The invention is not limited to the diagnosis andtreatment of so-called benign and malignant solid tumors, but theprinciples thereof have been found to also apply to haematologicalmalignancies like leukemias and lymphomas.

[0036] Recent publications indicate that atherosclerotic plaques alsoinvolve abnormal proliferation [26] of mainly smooth muscle cells and itwas postulated that atherosclerotic plaques constitute benign tumors[27]. Therefore, this type of disorder is also to be understood as apossible indication for the use of the MAG gene family, in particular indiagnostic and therapeutic applications.

[0037] As already indicated above it has been found that in certainmalignant tumors the expression level of the HMG genes is increased[28]. Until now the relevance of this observation was not understood.Another aspect of the invention thus relates to the implementation ofthe identification of the MAG genes in therapy. The invention forexample provides anti-sense molecules or expression inhibitors of theMAG gene for use in the treatment of diseases involving cells having anon-physiological proliferative capacity by modulating the expression ofthe gene. A non-physiological high expression may thus be normalised bymeans of antisense RNA that is either administered to the cell orexpressed thereby and binds to the mRNA, or antibodies directed to thegene product, which in turn may result in a normalisation of the cellgrowth. The examples will demonstrate that expression of antisense RNAin leukemic cells results in a re-differentiation of the cells back tonormal.

[0038] The invention thus provides derivatives of the MAG gene and/orits immediate environment for use in diagnosis and the preparation oftherapeutical compositions, wherein the derivatives are selected fromthe group consisting of sense and anti-sense cDNA or fragments thereof,transcripts of the gene or fragments thereof, antisense RNA, triplehelix inducing molecule or other types of “transcription clamps”,fragments of the gene or its complementary strand, proteins encoded bythe gene or fragments thereof, protein nucleic acids (PNA), antibodiesdirected to the gene, the cDNA, the transcript, the protein or thefragments thereof, as well as antibody fragments. Besides the use ofdirect derivatives of the genes and their surroundings (flankingsequences) in diagnosis and therapy, other molecules, like expressioninhibitors or expression enhancers, may be used for therapeutictreatment according to the invention. An example of this type ofmolecule are ribozymes that destroy RNA molecules.

[0039] Besides the above described therapeutic and diagnostic methodsthe principles of the invention may also be used for producing atransgenic animal model for testing pharmaceuticals for treatment of MAGgene related malignant or benign tumors and atherosclerotic plaques. Oneof the examples describes the production of such an animal model.

[0040] It is to be understood that the principles of the presentinvention are described herein for illustration purposes only withreference to the HMGI-C gene mapping at chromosome 12 and the HMGI(Y)gene mapping at chromosome 6 and the LPP gene on chromosome 3. Based onthe information provided in this application the skilled person will beable to isolate and sequence corresponding genes of the gene family andapply the principles of this invention by using the gene and itssequence without departing from the scope of the general concept of thisinvention.

[0041] The present invention will thus be further elucidated by thefollowing examples which are in no way intended to limit the scopethereof.

EXAMPLES Example 1

[0042] 1. Introduction

[0043] This example describes the isolation and analysis of 75overlapping YAC clones and the establishment of a YAC contig (set ofoverlapping clones), which spans about 6 Mb of genomic DNA around locusD12S8 and includes MAR. The orientation of the YAC contig on the longarm of chromosome 12 was determined by double-color FISH analysis. Onthe basis of STS-content mapping and restriction enzyme analysis, a longrange physical map of this 6 Mb DNA region was established. The contigrepresents a useful resource for cDNA capture aimed at identifying geneslocated in 12q15, including the one directly affected by the variouschromosome 12 aberrations.

[0044] 2. Materials and Methods

[0045] 2.1. Cell Lines

[0046] Cell-lines PK89-12 and LIS-3/SV40/A9-B4 were used for ChromosomeAssignment using Somatic cell Hybrids (CASH) experiments. PK89-12, whichcontains chromosome 12 as the sole human chromosome in a hamster geneticbackground, has been described before [29]. PK89-12 cells were grown inDME-F12 medium supplemented with 10% fetal bovine serum, 200 IU/mlpenicillin, and 200 μg/ml streptomycin. Somatic cell hybridLIS-3/SV40/A9-B4 was obtained upon fusion of myxoid liposarcoma cellline LIS-3/SV40, which carries a t(12;16) (q13;p11.2), and mouse A9cells and was previously shown to contain der(16), but neither der(12)nor the normal chromosome 12 [30]. LIS-3/SV40/A9-B4 cells were grown inselective AOA-medium (AOA-medium which consisted of DME-F12 mediumsupplemented with 10% fetal bovine serum, 0.05 mM adenine, 0.05 mMouabain, and 0.01 mM azaserine). Both cell lines were frequently assayedby standard cytogenetic techniques.

[0047] 2.2. Nucleotide Sequence Analysis and Oligonucleotides.

[0048] Nucleotide sequences were determined according to the dideoxychain termination method using a T7 polymerase sequencing kit(Pharmacia/LKB) or a dsDNA Cycle Sequencing System (GIBCO/BRL). DNAfragments were subcloned in pGEM-3Zf(+) and sequenced usingFITC-labelled standard SP6 or T7 primers, or specific primerssynthesized based upon newly obtained sequences. Sequencing results wereobtained using an Automated Laser Fluorescent (A.L.F.) DNA sequencer(Pharmacia Biotech) and standard 30 cm, 6% Hydrolink^(R), Long Range™gels (AT Biochem). The nucleotide sequences were analyzed using thesequence analysis software Genepro (Riverside Scientific), PC/Gene(IntelliGenetics), the IntelliGenetics Suite software package(IntelliGenetics, Inc.), and Oligo [31]. All oligonucleotides werepurchased from Pharmacia Biotech.

[0049] 2.3. Chromosome Preparations and Fluorescence In SituHybridization (FISH)

[0050] FISH analysis of YAC clones was performed to establish theirchromosomal positions and to identify chimeric clones. FISH analysis ofcosmid clones corresponding to STSs of YAC insert ends were performed toestablish their chromosomal positions. Cosmids were isolated from humangenomic library CMLW-25383 [32] or the arrayed chromosome 12-specificlibrary constructed at Lawrence Livermore National Laboratory (LL12NC01,ref. 33) according to standard procedures [34]. Routine FISH analysiswas performed essentially as described before [30, 35]. DNA was labelledwith biotin-11-dUTP (Boehringer) using the protocol of Kievits et al.[36]. Antifade medium, consisting of DABCO (2 g/100 ml, Sigma), 0.1 MTris-HCL pH 8, 0.02% Thimerosal, and glycerol (90%), and containingpropidium iodide (0.5 μg/ml, Sigma) as a counterstain, was added 15 minbefore specimens were analyzed on a Zeiss Axiophot fluorescencemicroscope using a double band-pass filter for FITC/Texas red (Omegaoptical, Inc.). Results were recorded on Scotch (3M) 640 ASA film.

[0051] For the double colour FISH experiments, LLNL12NC01-96C11 waslabelled with digoxygenin-11-dUTP (Boehringer) and cosmidsLLNL12NCO1-1F6 and -193F10, with biotin-11-dUTP. Equal amounts of eachprobe were combined and this mixture was used for hybridization. Afterhybridization, slides were incubated for 20 min with Avidin-FITC andthen washed as described by Kievits et al. [36]. Subsequent series ofincubations in TNB buffer (0.1 M Tris-HCl pH 7.5, 0.15 M NaCl, 0.5%Boehringer blocking agent (Boehringer)) and washing steps were performedin TNT buffer (0.1 M Tris-HCl pH 7.5, 0.15 M NaCl, 0.05%-Tween-20); allincubations were performed at 37° C. for 30 min. During the secondincubation, Goat-α-Avidin-biotin (Vector) and Mouse-α-digoxygenin(Sigma) were applied simultaneously. During the third incubation,Avidin-FITC and Rabbit-α-Mouse-TRITC (Sigma) were applied. During thelast incubation, Goat-α-Rabbit-TRITC (Sigma) was applied. After a lastwash in TNT buffer, samples were washed twice in 1× PBS and thendehydrated through an, ethanol series (70%, 90%, 100%). Antifade mediumcontaining 75 ng/μl DAPI (Serva) as counterstain was used. Specimenswere analyzed on a Zeiss Axiophot fluorescence microscope as describedabove.

[0052] 2.4. Screening of YAC Libraries.

[0053] YAC clones were isolated from CEPH human genomic YAC librariesmark 1 and 3 [37, 38] made available to us by the Centre d'Etude duPolyphormisme Humain (CEPH). Screening was carried out as previouslydescribed [39]. Contaminating Candida parapsylosis, which was sometimesencountered, was eradicated by adding terbinafin (kindly supplied by Dr.Dieter Römer, Sandoz Pharma LTD, Basle, Switzerland) to the growthmedium (final concentration: 25 μg/ml). The isolated YAC clones werecharacterized by STS-content mapping, contour-clamped homogeneouselectric field (CHEF) gel electrophoresis [40], restriction mapping, andhybridization- and FISH analysis.

[0054] 2.5. PCR Reactions

[0055] PCR amplification was carried out using a Pharmacia/LKB Gene ATAQController (Pharmacia/LKB) in final volumes of 100 μl containing 10 mMTris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatine, 2 mM dNTP's,20 pmole of each amplimer, 2.5 units of Amplitaq (Perkin-Elmer Cetus),and 100 ng (for superpools) or 20 ng (for pools) of DNA. After initialdenaturation for 5 min at 94° C., 35 amplification cycles were performedeach consisting of denaturation for 1 min at 94° C., annealing for 1 minat the appropriate temperature (see Table I) and extension for 1 min at72° C. The PCR reaction was completed by a final extension at 72° C. for5 min. Results were evaluated by analysis of 10 μl of the reactionproduct on polyacrylamide minigels.

[0056] 2.6. Pulsed-Field Gel Electrophoresis and Southern Blot Analysis

[0057] Pulsed-field gel electrophoresis and Southern blot analysis wereperformed exactly as described by Schoenmakers et al. [39]. Agaroseplugs containing high-molecular weight YAC DNA (equivalent to about1×10⁸ yeast cells) were twice equilibrated in approximately 25 ml TEbuffer (pH 8.0) for 30 min at 50° C. followed by two similar rounds ofequilibration at room temperature. Plugs were subsequently transferredto round-bottom 2 ml eppendorf tubes and equilibrated two times for 30min in 500 μl of the appropriate 1× restriction-buffer at theappropriate restriction temperature. Thereafter, DNA was digested in theplugs according to the suppliers (Boehringer) instructions for 4 h using30 units of restriction endonuclease per digestion reaction. Afterdigestion, plugs along with appropriate molecular weight markers wereloaded onto a 1% agarose/0.25×TBE gel, sealed with LMP-agarose and sizefractionated on a CHEF apparatus (Biorad) for 18 h at 6.0 V/cm using apulse angle of 120 degrees and constant pulse times varying from 10 sec(separation up to 300 kbp) to 20 sec (separation up to 500 kbp). In thecase of large restriction fragments, additional runs were performed,aiming at the separation of fragments with sizes above 500 kbp.Electrophoresis was performed at 14° C. in 0.25× TBE. As molecularweight markers, lambda ladders (Promega) and home-made plugs containinglambda DNA cut with restriction endonuclease HindIII were used. Afterelectrophoresis, gels were stained with ethidium bromide, photographed,and UV irradiated using a stratalinker (Stratagene) set at 120 mJ. DNAwas subsequently blotted onto Hybond N⁺ membranes (Amersham) for 4-16 husing 0.4 N NaOH as transfer buffer. After blotting, the membranes weredried for 15 min at 80° C., briefly neutralised in 2× SSPE, andprehybridised for at least 3 h at 42° C. in 50 ml of a solutionconsisting of 50% formamide, 5× SSPE, 5× Denhardts, 0.1% SDS and 200μg/ml heparin. Filters were subsequently hybridised for 16 h at 42° C.in 10 ml of a solution consisting of 50% formamide, 5× SSPE, 1×Denhardts, 0.1% SDS, 100 μg/ml heparin, 0.5% dextran sulphate and2-3×10⁶ cpm/ml of labelled probe. Thereafter, membranes were firstwashed two times for 5 min in 2× SSPE/0.1% SDS at room temperature, thenfor 30 min in 2× SSPE/0.1% SDS at 42° C. and, finally, in 0.1× SSPE/0.1%SDS for 20 min at 65° C. Kodak XAR-5 films were exposed at −80° C. for3-16 h, depending on probe performance. Intensifying screens (Kyokkospecial 500) were used.

[0058] 2.7. Generation of STSs From YAC Insert Ends

[0059] STSs from YAC insert ends were obtained using a vectorette-PCRprocedure in combination with direct DNA sequencing analysis,essentially as described by Geurts et al. [41]. Primer sets weredeveloped and tested on human genomic DNA, basically according toprocedures described above. STSs will be referred to throughout thisapplication by their abbreviated names (for instance: RM1 instead of STS12-RM1) for reasons of convenience.

[0060] 3. Results

[0061] 3.1. Assembly of a YAC Contig Around Locus D12S8

[0062] In previous studies [39], a 800 kb YAC contig around D12S8 wasdescribed. This contig consisted of the following three partiallyoverlapping, non-chimeric CEPH YAC clones: 258F11, 320F6, and 234G11.This contig was used as starting point for a chromosome walking projectto define the DNA region on the long arm of chromosome 12 thatencompasses the breakpoints of a variety of benign solid tumors, whichare all located proximal to D12S8 and distal to CHOP. Initially,chromosome walking was performed bidirectionally until the size of thecontig allowed reliable determination of the orientation of it. In thebidirectional and subsequent unidirectional chromosome walking steps,the following general procedure was used. First, rescuing and sequencingthe ends of YAC clones resulted in DNA markers characterizing the leftand right sides of these (Table I). Based on sequence data of the endsof forty YAC inserts, primer sets were developed for specificamplification of DNA; establishing STSs (Table II). Their localizationto 12q13-qter was determined by CASH as well as FISH after correspondingcosmid clones were isolated. It should be noted that isolated YAC cloneswere often evaluated by FISH analysis too, thus not only revealing thechromosomal origin of their inserts but also, for a number of cases,establishing and defining their chimeric nature. Moreover, it should beemphasized that data obtained by restriction endonuclease analysis ofoverlapping YAC clones were also taken into account in the YAC cloneevaluation and subsequent alignment. With the sequentially selected andevaluated primer sets, screening of the YAC and cosmid libraries wasperformed to isolate the building blocks for contig-assembly. Therefore,contig-assembly was performed using data derived from FISH- andSTS-content mapping as well as restriction endonuclease analysis. Usingthis approach, we established a YAC contig consisting of 75 overlappingYAC clones, covering approximately 6 Mb of DNA (FIG. 1). This contigappeared to encompass the chromosome 12 breakpoints of all tumor-derivedcell lines studied [39]. Characteristics of the YACs that were used tobuild this contig are given in Table I.

[0063] 3.2. Establishment of the Chromosomal Orientation of the YACContig

[0064] To allow unidirectional chromosome walking towards the centromereof chromosome 12, the orientation of the DNA region flanked by STSs RM14and RM26 (approximate size: 1450 kb) was determined by double-colorinterphase FISH analysis. Cosmid clones corresponding to these STSs(i.e. LL12NC01-1F6 (RM14) and LL12NC01-96C11 (RM26)) were differentiallylabelled to show green or red signals, respectively. As a referencelocus, cosmid LL12NC01-193F10 was labelled to show green signals upondetection. LL12NC01-193F10 had previously been mapped distal to thebreakpoint of LIS-3/SV40 (i.e. CHOP) and proximal to the chromosome 12qbreakpoints in lipoma cell line Li-14/SV40 and uterine leiomyoma cellline LM-30.1/SV40. LL12NC01-1F6 and LL12NC01-96C11 were found to bemapping distal to the 12q breakpoints in lipoma cell line Li-14/SV40 anduterine leiomyoma cell line LM-30.1/SV40. Therefore, LL12NC01-193F10 wasconcluded to be mapping proximal to both RM14 and RM26 (unpublishedresults). Of 150 informative interphases scored, 18% showed asignal-order of red-green-green whereas 72% showed a signal order ofgreen-red-green. Based upon these observations, we concluded that RM26mapped proximal to RM14, and therefore we continued to extend the YACcontig from the RM26 (i.e. proximal) side of our contig only. Only thecosmids containing RM14 and RM26 were ordered by double-color interphasemapping; the order of all others was deduced from data of the YACcontig. Finally, it should be noted that the chromosomal orientation ofthe contig as proposed on the basis of results of the double-colorinterphase FISH studies was independently confirmed after the YAC contighad been extended across the chromosome 12 breakpoints as present in avariety of tumor cell lines. This confirmatory information was obtainedin extensive FISH studies in which the positions of YAC and cosmidclones were determined relative to the chromosome 12q13-q15 breakpointsof primary lipomas, uterine leiomyomas, pleomorphic salivary glandadenomas, and pulmonary chondroid hamartomas or derivative cell lines[24, 42, 25, 43].

[0065] 3.3. Construction of a Rare-Cutter Physical Map from the 6 Mb YACContig around D12S8

[0066] Southern blots of total yeast plus YAC DNA, digested tocompletion with rare-cutter enzymes (see Materials and Methods) andseparated on CHEF gels, were hybridized sequentially with i) the STSused for the initial screening of the YAC in question, ii) pYAC4 rightarm sequences, iii) pYAC4 left arm sequences, and iv) a human ALU-repeatprobe (BLUR-8). The long-range restriction map that was obtained in thisway, was completed by probing with PCR-isolated STSs/YAC end probes.Occasionally double-digests were performed.

[0067] Restriction maps of individual YAC clones were aligned and aconsensus restriction map was established. It is important to note herethat the entire consensus rare-cutter map was supported by at least twoindependent clones showing a full internal consistency.

[0068] 3.4. Physical Mapping of CA Repeats and Monomorphic STSs/ESTs

[0069] Based upon integrated mapping data as emerged from the SecondInternational Workshop on Human Chromosome 12 [44], a number ofpublished markers was expected to be mapping within the YAC contigpresented here. To allow full integration of our mapping data with thoseobtained by others, a number of markers were STS content-mapped on ourcontig, and the ones found positive were subsequently sublocalized by(primer-)hybridization on YAC Southern blots. Among the markers thatwere found to reside within the contig presented here were CA repeatsD12S313 (AFM207xf2) and D12S335 (AFM273vg9) [45], D12S375 (CHLCGATA3F02), and D12S56 [46]. Furthermore, the interferon gamma gene(IFNG) [47], the ras-related protein gene Rap1B [48], and expressedsequence tag EST01096 [49] were mapped using primer sets which wedeveloped based on publicly available sequence data (see Table II).Markers which were tested and found negative included D12S80(AFM102xd6), D12S92 (AFM203va7), D12S329 (AFM249xh9) and D12S344(AFM296xd9).

[0070] 4. Discussion

[0071] In the present example the establishment of a YAC contig andrare-cutter physical map covering approximately 6 Mb on 12q15, a regionon the long arm of human chromosome 12 containing MAR in which a numberof recurrent chromosomal aberrations of benign solid tumors are known tobe mapping was illustrated.

[0072] The extent of overlap between individual YACs has been carefullydetermined, placing the total length of the contig at approximately 6 Mb(FIG. 1). It should be noted that our sizing-data for some of the YACclones differ slightly from the sizes determined by CEPH [50]. It is ourbelief that this is most probably due to differences in the parametersfor running the pulsed-field gels in the different laboratories.

[0073] Using restriction mapping and STS-content analysis, a consensuslong range physical map (FIG. 1) was constructed. The entire compositemap is supported by at least two-fold coverage. In total over 30 Mb ofYAC DNA was characterized by restriction and STS content analysiscorresponding to an average contig coverage of about 5 times. Althoughthe “inborn” limited resolution associated with the technique ofpulsed-field electrophoresis does not allow very precise sizeestimations, comparison to restriction mapping data obtained from a 500kb cosmid contig contained within the YAC contig presented here showed aremarkable good correlation. Extrapolating from the cosmid data, weestimate the accuracy of the rare-cutter physical map presented here atabout 10 kb.

[0074] The results of our physical mapping studies allowed integrationof three gene-specific as well as five anonymous markers isolated byothers (indicated in between arrows in FIG. 1). The anonymous markersinclude one monomorphic and four polymorphic markers. Five previouslypublished YAC-end-derived single copy STSs (RM1, RM4, RM5, RM7, andRM21) as well as four published CA repeats (D12S56, D12S313, D12S335,and D12S375) and three published gene-associated STSs/ESTs (RAP1B,EST01096, and IFNG) have been placed on the same physical map and thiswill facilitate (linkage-) mapping and identification of a number oftraits/disease genes that map in the region. Furthermore, we were ableto place onto the same physical map, seventy two YAC-end-derived (TableI) and eight cosmid-end- or inter-ALU-derived DNA markers (CH9, RM1,RM110, RM111, RM130, RM131, RM132, and RM133), which were developedduring the process of chromosome walking. The PYTHIA automatic mailserver at PYTHIA@anl.gov was used to screen the derived sequences ofthese DNA markers for the presence of repeats. For forty three of theseseventy two DNA markers (listed in Table II), primer sets were developedand the corresponding STSs were determined to be single copy by PCR aswell as Southern blot analysis of human genomic DNA. The twenty nineremaining DNA markers (depicted in the yellow boxes) representYAC-end-derived sequences for which we did not develop primer sets.These YAC-end sequences are assumed to be mapping to chromosome 12 onthe basis of restriction mapping. The final picture reveals an overallmarker density in this region of approximately one within every 70 kb.

[0075] The analysis of the contig presented here revealed many CpG-richregions, potentially HTF islands, which are known to be frequentlyassociated with housekeeping genes. These CpG islands are most probablylocated at the 5′ ends of as yet unidentified genes: it has been shownthat in 90% of cases in which three or more rare-cutter restrictionsites coincide in YAC DNA there is an associated gene [51]. This islikely to be an underestimate of the number of genes yet to beidentified in this region because 60% of tissue-specific genes are notassociated with CpG islands [52] and also because it is possible for twogenes to be transcribed in different orientations from a single island[53].

[0076] While several of the YAC clones that were isolated from the CEPHYAC library mark 1 were found to be chimeric, overlapping YAC clonesthat appeared to be non-chimeric based on FISH, restriction mapping andSTS content analysis could be obtained in each screening, which is inagreement with the reported complexity of the library. The degree ofchimerism for the CEPH YAC library mark 1 was determined at 18% (12 outof 68) for the region under investigation here. The small number of YACsfrom the CEPH YAC library mark 3 (only 7 MEGA YACs were included in thisstudy) did not allow a reliable estimation of the percentage of chimericclones present in this library. The average size of YACs derived fromthe mark 1 library was calculated to be 381 kb; non-chimeric YACs (n=58)had an average size of 366 kb while chimeric YACs (n=12) were found tohave a considerable larger average size; i.e. 454 kb.

[0077] In summary, we present a 6 Mb YAC contig corresponding to a humanchromosomal region which is frequently rearranged in a variety of benignsolid tumors. The contig links over 84 loci, including 3 gene-associatedSTSs. Moreover, by restriction mapping we have identified at least 12CpG islands which might be indicative for genes residing there. Finally,four CA repeats have been sublocalized within the contig. Theintegration of the genetic, physical, and transcriptional maps of theregion provides a basic framework for further studies of this region ofchromosome 12. Initial studies are likely to focus on MAR and ULCR12, asthese regions contain the breakpoint cluster regions of at least threedistinct types of solid tumors. The various YAC clones we describe hereare valuable resources for such studies. They should facilitate thesearch for genes residing in this area and the identification of thosedirectly affected by the chromosome 12q aberrations of the variousbenign solid tumors.

Example 2

[0078] 1. Introduction

[0079] It was found that the 1.7 Mb Multiple Aberration Region on humanchromosome 12q15 harbors recurrent chromosome 12 breakpoints frequentlyfound in different benign solid tumor types. In this example theidentification of an HMG gene within MAR that appears to be ofpathogenetical relevance is described. Using a positional cloningapproach, the High Mobility Group protein gene HMGI-C was identifiedwithin a 175 kb segment of MAR and its genomic organizationcharacterized. By FISH, within this gene the majority of the breakpointsof seven different benign solid tumor types were pinpointed. By Southernblot and 3′-RACE analysis, consistent rearrangements in HMGI-C and/orexpression of altered HMGI-C transcripts were demonstrated. Theseresults indicate a link between a member of the HMG gene family andbenign solid tumor development.

[0080] 2. Materials and Methods

[0081] 2.1. Cell Culture and Primary Tumor Specimens.

[0082] Tumor cell lines listed in FIG. 3 were established by atransfection procedure [54] and have been described before in [39, 24]and in an article of Van de Ven et al., Genes Chromosom. Cancer 12,296-303 (1995) enclosed with this application as ANNEX 1. Cells weregrown in TC199 medium supplemented with 20% fetal bovine serum and wereassayed by standard cytogenetic techniques at regular intervals. Thehuman hepatocellular carcinoma cell lines Hep 3B and Hep G2 wereobtained from the ATCC (accession numbers ATCC HB 8064 and ATCC HB 8065)and cultured in DMEM/F12 supplemented with 4% Ultroser (Gibco/BRL).Primary solid tumors were obtained from various University Clinics.

[0083] 2.2. YAC and Cosmid Clones

[0084] YAC clones were isolated from the CEPH mark 1 [57] and mark 3[58] YAC libraries using a combination of PCR-based screening [59] andcolony hybridization analysis. Cosmid clones were isolated from anarrayed human chromosome 12-specific cosmid library (LL12NC01) [60]obtained from Lawrence Livermore National Laboratory (courtesy P. deJong). LL12NC01-derived cosmid clones are indicated by their microtiterplate addresses; i.e. for instance 27E12.

[0085] Cosmid DNA was extracted using standard techniques involvingpurification over Qiagen tips (Diagen). Agarose plugs containinghigh-molecular weight yeast+YAC DNA (equivalent to 1×10⁹ cells ml⁻¹)were prepared as described before [61]. Plugs were thoroughly dialysedagainst four changes of 25 ml T₁₀E₁ (pH 8.0) followed by two changes of0.5 ml 1× restriction buffer before they were subjected to eitherpulsed-field restriction enzyme mapping or YAC-end rescue. YAC-endrescue was performed using a vectorette-PCR procedure in combinationwith direct solid phase DNA sequencing, as described before in reference61. Inter-Alu PCR products were isolated using publishedoligonucleotides TC65 or 517 [62] to which SalI-tails were added tofacilitate cloning. After sequence analysis, primer pairs were developedusing the OLIGO computer algorithm [61].

[0086] 2.3. DNA Labelling

[0087] DNA from YACs, cosmids, PCR products and oligonucleotides waslabelled using a variety of techniques. For FISH, cosmid clones orinter-Alu PCR products of YACs were biotinylated with biotin-11-dUTP(Boehringer) by nick translation. For filter hybridizations, probes wereradio-labelled with α-³²P-dCTP using random hexamers [62]. In case ofPCR-products smaller than 200 bp in size, a similar protocol wasapplied, but specific oligonucleotides were used to prime labellingreactions. Oligonucleotides were labelled using γ-³²P-ATP.

[0088] 2.4. Nucleotide Sequence Analysis and PCR Amplification

[0089] Nucleotide sequences were determined as described in Example 1.Sequencing results were analyzed using an A.L.F. DNA sequencer™(Pharmacia Biotech) on standard 30 cm, 6% Hydrolink^(R), Long Range™gels (AT Biochem). PCR amplifications were carried out essentially asdescribed before [39].

[0090] 2.5. Rapid Amplification of cDNA Ends (RACE)

[0091] Rapid amplification of 3′ cDNA-ends (3′-RACE) was performed usinga slight modification of part of the GIBCO/BRL 3′-ET protocol. For firststrand cDNA synthesis, adapter primer (AP2) AAG GAT CCG TCG ACA TC(T)₁₇was used. For both initial and secondary rounds of PCR, the universalamplification primer (UAP2) CUA CUA CUA CUA AAG GAT CCG TCG ACA TC wasused as “reversed primer”. In the first PCR round the following specific“forward primers” were used: i) 5′-CTT CAG CCC AGG GAC AAC-3′. (exon 1),ii) 5′-CAA GAG GCA GAC CTA GGA-3′ (exon 3), or iii) 5′-AAC AAT GCA ACTTTT AAT TAC TG-3′ (3′-UTR). In the second PCR round the followingspecific forward primers (nested primers as compared to those used inthe first round) were used: i) 5′-CAU CAU CAU CAU CGC CTC AGA AGA GAGGAC-3′ (exon 1), ii) 5′-CAU CAU CAU CAU GTT CAG AAG AAG CCT GCT-3′ (exon4), or iii) 5′-CAU CAU CAU CAU TTG ATC TGA TAA GCA AGA GTG GG-3′(3′-UTR). CUA/CAU-tailing of the nested, specific primers allowed theuse of the directional CloneAmp cloning system (GIBCO/BRL).

[0092] 3. Results

[0093] 3.1. Development of Cosmid Contig and STS Map of MAR Segment

[0094] During the course of a positional cloning effort focusing on thelong arm of human chromosome 12, we constructed a yeast artificialchromosome (YAC) contig spanning about 6 Mb and consisting of 75overlapping YACs. For a description thereof reference is made toExample 1. This contig encompasses MAR (see also FIG. 2), in which mostof the chromosome 12q13-q15 breakpoints as present in a variety ofprimary benign solid tumors (34 tumors of eight different types testedsofar; Table 5) and tumor cell lines (26 tested sofar, derived fromlipoma, uterine leiomyoma, and pleomorphic salivary gland adenoma; FIG.3) appear to cluster. We have developed both a long-range STS and rarecutter physical map of MAR and found, by FISH analysis, most of thebreakpoints mapping within the 445 kb subregion of MAR located betweenSTSs RM33 and RM98 (see FIG. 2 and 3). FISH experiments, includingextensive quality control, were performed according to routineprocedures as described before [25, 39, 24, 42, 36] To further refinethe distribution of breakpoints within this 445 kb MAR segment, a cosmidcontig consisting of 54 overlapping cosmid clones has been developed anda dense STS map (FIG. 2) established. The cosmid contig wasdouble-checked by comparison to the rare cutter physical map and by STScontent mapping.

[0095] 3.2. Clustering of the Chromosome 12q Breakpoints Within a 175 kbDNA Segment of MAR

[0096] The chromosome 12q breakpoints in the various tumor cell linesstudied was pinpointed within the cosmid contig by FISH (FIG. 3). Aspart of our quality control FISH experiments [25, 39, 24, 42], selectedcosmids were first tested on metaphase spreads derived from normallymphocytes. FISH results indicated that the majority (at least 18 outof the 26 cases) of the chromosome 12 breakpoints in these tumor celllines were found to be clustering within the 175 kb DNA interval betweenRM99 and RM133, indicating this interval to constitute the mainbreakpoint cluster region. FISH results obtained with Li-501/SV40indicated that part of MAR was translocated to an apparently normalchromosome 3; a chromosome aberration overseen by applied cytogenetics.Of interest to note, finally, is the fact that the breakpoints ofuterine leiomyoma cell lines LM-5.1/SV40, LM-65/SV40, and LM-608/SV40were found to be mapping within the same cosmid clone; i.e. cosmid27E12.

[0097] We also performed FISH experiments on eight different types ofprimary benign solid tumors with chromosome 12q13-q15 aberrations (Table4). A mixture of cosmid clones 27E12-and 142H1 was used as molecularprobe. In summary, the results of the FISH studies of primary tumorswere consistent with those obtained for the tumor cell lines. Theobservation that breakpoints of each of the seven different tumor typestested were found within the same 175 kb DNA interval of MAR suggestedthat this interval is critically relevant to the development of thesetumors and, therefore, might harbor the putative MAG locus or criticalpart(s) of it.

[0098] 3.3. Identification of Candidate Genes Mapping Within MAR

[0099] In an attempt to identify candidate genes mapping within the 175kb subregion of MAR between STSs RM99 and RM133, we used 3′-terminalexon trapping and genomic sequence sampling (GSS) [63]. Using the GSSapproach, we obtained DNA sequence data of the termini of a 4.9 kb BamHIsubfragment of cosmid 27E12, which was shown by FISH analysis to besplit by the chromosome 12 aberrations in three of the uterine leiomyomacell lines tested. A BLAST [64] search revealed that part of thesesequences displayed sequence identity with a publicly available partialcDNA sequence (EMBL accession #. Z31595) of the high mobility group(HMG) protein gene HMGI-C [65], which is a member of the HMG gene family[66]. In light of these observations, HMGI-C was considered a candidateMAG gene and studied in further detail.

[0100] 3.4. Genomic Organization of HMGI-C and Rearrangements in BenignSolid Tumors

[0101] Since only 1200 nucleotides of the HMGI-C transcript (reportedsize approximately 4 kb [65, 67]) were publicly available, we firstdetermined most of the remaining nucleotide sequences of the HMGI-Ctranscript (GenBank, # U28749). This allowed us to subsequentlyestablish the genomic organization of the gene. Of interest to noteabout the sequence data is that a CT-repeat is present in the 5′-UTR ofHMGI-C and a GGGGT-pentanucleotide repeat in the 3′-UTR, which might beof regulatory relevance. Comparison of transcribed to genomic DNAsequences (GenBank, # U28750, U28751, U28752, U28753, and U28754) of thegene revealed that HMGI-C contains at least 5 exons (FIG. 2).Transcriptional orientation of the gene is directed towards the telomereof the long arm of the chromosome. Each of the first three exons encodea putative DNA binding domain (DBD), and exon 5 encodes an acidicdomain, which is separated from the three DBDs by a spacer domainencoded by exon 4. The three DBD-encoding exons are positionedrelatively close together and are separated by a large intron of about140 kb from the two other exons, which in turn are separated about 11 kbfrom each other. Of particular interest to emphasize here is that thefive exons are dispersed over a genomic region of at least 160 kb, thusalmost covering the entire 175 kb main MAR breakpoint cluster regiondescribed above. Results of molecular cytogenetic studies, using amixture of cosmids 142H1 (containing exons 1-3) and 27E12 (containingexons 4 and 5) as molecular probe, clearly demonstrate that the HMGI-Cgene is directly affected by the observed chromosome 12 aberrations inthe majority of the tumors and tumor cell lines that were evaluated(FIG. 3; Table 4). These cytogenetic observations were independentlyconfirmed by Southern blot analysis in the case of LM-608/SV40 (resultsnot shown) LM-30.1/SV40 [24], and Ad-312/SV40; probes used includedCH76, RM118-A, and EM26. The failure to detect the breakpoints ofLM-65/SV40, LM-609/SV40, Ad-211/SV40, Ad-263/SV40, Ad-302/SV40,Li-14/SV40, and Li-538/SV40 with any of these three probes was alsoconsistent with the FISH data establishing the relative positions of thebreakpoints in MAR (cf. FIG. 3). These results made HMGI-C a primecandidate to be the postulated MAG gene.

[0102] 3.5. Expression of Aberrant HMGI-C Transcripts in Benign SolidTumor Cells.

[0103] In the context of follow-up studies, it was of interest to testfor possible aberrant HMGI-C expression. Initial Northern blot studiesrevealed that transcripts of HMGI-C could not be detected in a varietyof normal tissues (brain, heart, lung, liver, kidney, pancreas,placenta, skeletal muscle) tested as well as in a number of the tumorcell lines listed in FIG. 3 (data not shown). It is known that HMGI-CmRNA levels in normal differentiated tissues are very much lower than inmalignant tissues [65, 67]. As a control in our Northern studies, weincluded hepatoma cell line Hep 3B, which is known to express relativelyhigh levels of HMGI-C. We readily detected two major HMGI-C transcripts,approximately 3.6 and 3.2 kb in size; with the differences in molecularweight most likely due to differences in their 5′-noncoding regions. Inan alternative and more sensitive approach to detect HMGI-C or3′-aberrant HMGI-C transcripts, we have performed 3′-RACE experiments.In control experiments using a number of tissues with varying HMGI-Ctranscription levels (high levels in Hep 3B hepatoma cells, intermediatein Hep G2 hepatoma cells, and low in myometrium, normal fat tissue, andpseudomyxoma), we obtained 3′-RACE clones which, upon molecular cloningand nucleotide sequence analysis, appeared to represent perfect partialcDNA copies of 3′-HMGI-C mRNA sequences; no matter which of the threeselected primer sets was used (see Methodology). RACE products mostlikely corresponding to cryptic or aberrantly spliced HMGI-C transcriptswere occasionally observed; their ectopic sequences were mapped back toHMGI-C intron 3 or 4.

[0104] In similar 3′-RACE analysis of ten different primary tumors ortumor cell lines derived from lipoma, uterine leiomyoma, and pleomorphicsalivary gland adenoma, we detected both constant and unique PCRproducts. The constant PCR products appeared to represent, in mostcases, perfect partial cDNA copies of 3′-HMGI-C mRNA sequences. Theymost likely originated from a presumably unaffected HMGI-C allele andmight be considered as internal controls. The unique PCR products of theten tumor cell samples presented here appeared to contain ectopicsequences fused to HMGI-C sequences. In most cases, the ectopicsequences were found to be derived from the established translocationpartners, thus providing independent evidence for translocation-inducedrearrangements of the HMGI-C gene. Information concerning nucleotidesequences, diversion points, and chromosomal origins of the ectopicsequences of these RACE products is summarized in Table 5. It should benoted that chromosomal origins of ectopic sequences was established byCASH (Chromosome Assignment using Somatic cell Hybrids) analysis usingthe NIGMS Human/Rodent Somatic Hybrid Mapping Panel 2 obtained from theCoriell Cell Repositories. Chromosomal assignment was independentlyconfirmed by additional data for cases pCH1111, pCH172, pCH174, pCH193,and pCH117, as further outlined in Table 5. Taking into account thelimitations of conventional cytogenetic analysis, especially in caseswith complex karyotypes, the chromosome assignments of the ectopicsequences are in good agreement with the previous cytogeneticdescription of the translocations.

[0105] Somewhat unexpected were the data obtained with Ad-312/SV40, asavailable molecular cytogenetic analysis had indicated its chromosome 12breakpoint to map far outside the HMGI-C gene; over 1 Mb [42]. Theectopic sequences appeared to originate from chromosome 1 (moreprecisely from a segment within M.I.T. YAC contig WC-511, which ispartially mapping at 1p22), the established translocation partner (FIG.2). Further molecular analysis is required to precisely define theeffect on functional expression of the aberrant HMGI-C gene in thisparticular case. Of further interest to note here, is that the sequencescoming from chromosome 1 apparently remove the GGGGT repeat observed inthe 3′-UTR region of HMGI-C, as this repeat is not present in the RACEproduct. In contrast, in primary uterine leiomyoma LM-#58(t(8;12)(q24;q14-q15)), which was shown to have its breakpoint also inthe 3′-UTR, this repeat appeared to be present in the RACE product.Therefore, removal of this repeat is most probably not critical fortumor development. The results with primary tumor LM-#168.1, in whichthe X chromosome is the cytogenetically assigned translocation partner,revealed that the ectopic sequences were derived from chromosome 14; thepreferential translocation partner in leiomyoma. It is possible thatinvolvement of chromosome 14 cannot be detected by standard karyotypingin this particular case, as turned out to be the case for Li-501/SV40.In primary lipoma Li-#294 (t(8;12)(q22;q14)), two alternative ectopicsequences were detected. Additional CASH analysis using a hybrid cellmapping panel for regional localization of probes to human chromosome 8[68] revealed that these were both derived from chromosome 8q22-qter(Table 5). It is very well possible that these RACE products correspondto alternatively spliced transcripts. Finally, in four of the cases(Table 5, cases pCH114, pCH110, pCH109, pCH116), the RACE productsappeared to correspond to cryptic or aberrantly spliced HMGI-Ctranscripts, as the corresponding ectopic sequences were found to bederived from either HMGI-C intron 3 or 4. Such RACE products have alsobeen observed in the control experiments described above. In conclusion,the detection of aberrant HMGI-C transcripts in the tumor cells providesadditional strong support of HMGI-C being consistently rearranged by thevarious chromosome 12 aberrations. It should be noted that the aberrantHMGI-C transcripts in the various cases should be characterized in fulllength before any final conclusion can be drawn about biologicalimplications.

[0106] A first and preliminary evaluation of isolated ectopic sequencesrevealed in phase open reading frames of variable length. In the case ofprimary tumor LM-#25, for instance, already the second codon in theectopic sequences appeared to be a stop codon (Table 5). A note ofcaution is appropriate here, as sequence data have been obtained onlyfor clones that were produced via two rounds of extensive (probablymutations inducing) PCR. For Li-501/SV40, it is of interest to notethat, in Northern blot analysis, the isolated ectopic sequences detecteda transcript of over 10 kb in a variety of tissues, including heart,kidney, liver, lung, pancreas, placenta, and skeletal muscle, but not inbrain (data not shown). As chromosome 3 is the preferred partner in thechromosome 12q13-q15 translocations in lipomas and the chromosome 3breakpoints of various lipomas were found to be spanned by YAC cloneCEPH192B10, the detected transcript might correspond to a putativelipoma-preferred partner gene (LPP).

[0107] 4. Discussion

[0108] In ANNEX 1 it was demonstrated that the chromosome 12q13-q15breakpoints of lipoma, pleomorphic salivary gland adenoma, and uterineleiomyoma, irrespective of their cytogenetic assignments in the past tosegment q13, q14, or q15 of chromosome 12, all cluster within the 1.7 MbDNA interval designated MAR. In support of the claimed clustering ofbreakpoints is a recent study by Schoenberg Fejzo et al. [14],identifying a CEPH mega-YAC spanning the chromosome 12 translocationbreakpoints in two of the three tumor types. In the present study, wehave conclusively demonstrated by FISH analysis that chromosome 12breakpoints of seven different solid tumor types are clustering within arelatively small (175 kb) segment of MAR. For some tumor cell lines,Southern blot data were obtained and these were always in support of theFISH results. From all these observations, we conclude that this segmentof MAR constitutes a major target area for the chromosome 12 aberrationsin these tumors and that it is likely to represent the postulated MAGlocus: the multi-tumor aberrant growth locus that might be considered ascommon denominator in these tumors.

[0109] Within the 175 kb MAR segment, we have identified the HMGI-C geneand determined characteristics of its genomic organization.Structurally, the HMGI-C-encoded phosphoprotein consists of threeputative DNA binding domains, a spacer region, and an acidiccarboxy-terminal domain, and contains potential sites of phosphorylationfor both casein kinase II and p34/cdc2 [65, 67]. We have provided strongevidence that HMGI-C is a prime candidate target gene involved in thevarious tumor types studied here. In FISH studies, the breakpoints of 29out of 33 primary tumors were found to be mapping between two highlyinformative cosmids 142H1 and 27E12; the first one containing the threeDBD-encoding exons and the second one, the remaining exons that code forthe two other domains. Therefore, the majority of the breakpoints mapwithin the gene, most of them probably within the 140 kb intron (intron3), which is also in line with FISH results obtained with the 26 tumorcell lines that were evaluated. It should also be noted that the 5′-endof the HMGI-C gene is not yet fully characterized. As HMGI (Y), anothermember of this gene family, is known to possess various alternativefirst exons [69], the size of the HMGI-C gene might be larger than yetassumed. Further support that HMGI-C is affected by the chromosome 12aberrations can be deduced from the results of the 3′-RACE experiments.Aberrant HMGI-C transcripts were detected in tumor cells, consisting oftranscribed HMGI-C sequences fused to newly acquired sequences, in mostcases clearly originating from the chromosomes that were cytogeneticallyidentified as the translocation partners. It is noteworthy that manychromosomes have been found as translocation partner in the tumorsstudied. This observed heterogeneity in the reciprocal breakpointregions involved in these translocations resembles that of a variety ofhematological malignancies with chromosome 11q23 rearrangementsinvolving the MLL gene [70], the translational product of which carriesan amino-terminal motif related to the DNA-binding motifs of HMGIproteins.

[0110] An intriguing issue pertains to the effect of the chromosome 12aberrations on expression of the HMGI-C gene and the directphysiological implications. Some functional characteristics of HMGI-Care known or may be deduced speculatively from studies of other familymembers. As it binds in the minor groove of DNA, it has been suggestedthat HMGI-C may play a role in organising satellite chromatin or act asa transcription factor [71, 72]. Studies on HMGI(Y), which is the membermost closely related to HMGI-C, have suggested that HMGI(Y) may functionas a promoter-specific accessory factor for NF-κ B transcriptionalactivity [73]. HMGI(Y) has also been shown to stimulate or inhibit DNAbinding of distinct transcriptional factor ATF-2 isoforms [74]. Bothstudies indicate that the protein may simply constitute a structuralcomponent of the transcriptional apparatus functioning inpromoter/enhancer contexts. In a recent report on high mobility groupprotein 1 (HMG1), yet another member of the HMG gene family with asimilar domain structure as HMGI-C and acting as a quasi-transcriptionfactor in gene transcription, a truncated HMG1 protein lacking theacidic carboxy-terminal region was shown to inhibit gene transcription[75]. It was put forward that the acidic terminus of the HMG1 moleculeis essential for the enhancement of gene expression in addition toelimination of the repression caused by the DNA binding. As most of thechromosome 12 breakpoints seem to occur in the 140 kb intron, separationof the DBDs from the acidic carboxy-terminal domain seems to occurfrequently. In case the acidic domain in HMGI-C has a similar functionas the one in HGMI(Y), the result of the chromosome 12 aberrations islikely to affect gene expression. Finally, it should be noted that thefate of the sequences encoding the acidic carboxy-terminal region is notyet known.

[0111] As HMGI-C is the first member of the HMG gene family that mightbe implicated in the development of benign tumors, the question arisesas to whether other members of this family could also be involved. TheHMG protein family consists of three subfamilies: i) the HMG1 and 2 typeproteins, which have been found to enhance transcription in vitro andmay well be members of a much larger class of regulators with HMG boxes;ii) the random-coil proteins HMG14 and 17 with an as yet unknownfunction; iii) the HMGI-type proteins, which bind to-the minor grooveand include HMGI-C, HMGI, and HMGI-Y; the latter two are encoded by thesame gene. It is of interest to note that published mapping positions ofmembers of the HMG family coincide with published chromosome breakpointsof benign solid tumors such as those studied here. The HMGI(Y) gene, forinstance, has been mapped to human chromosome 6p21 [69], which is knownto be involved in recurrent translocations observed in uterineleiomyoma, lipoma, and pleomorphic salivary gland adenoma [76]. Aslisted in the Human Genome Data Base, not all known members of the HMGfamily have been chromosomally assigned yet, although for some of them arelatively precise mapping position has been established. For instance,HMG17 to chromosome 1p36.1-p35, HMG1L to 13q12, and HMG14 to 21q22.3;all chromosome segments in which chromosome breakpoints of the tumortypes studied here have been reported [76]. Whether HMGI(Y) or any otherof these HMG members are indeed affected in other subgroups of thesetumors remains to be established. Of interest to mention, furthermore,are syndromes such as Bannayan-Zonana (McKusick #153480); Proteus(McKusick #176920), and Cowden (McKusick #158350); the latter syndromeis also called multiple hamartoma syndrome. In 60% of the individualswith congenital Bannayan-Zonana syndrome, a familial macrocephaly withmesodermal hamartomas, discrete lipomas and hemangiomas were found [70].

[0112] Finally, one aspect of our results should not escape attention.All the tumors that were evaluated in this study were of mesenchymalorigin or contained mesenchymal components. It would be of greatinterest to find out whether the observed involvement of HMGI-C ismesenchyme-specific or may be found also in tumors of non-mesenchymalorigin. The various DNA clones we describe here are valuable resourcesto address this important issue and should facilitate studies toconclusively implicate the HMGI-C gene in tumorigenesis.

Example 3

[0113] Rearrangements of Another Member of the HMG Gene Family

[0114] 1. Introduction

[0115] This example clearly demonstrates that within a given tumorentity (e.g. pulmonary chondroid hamartomas, uterine leiomyomas,endometrial polyps) tumors, histologically practically indistinguishablefrom each other, arise if either the HMGI-C gene or the HMGI(Y) gene isaffected by chromosomal rearrangements. Thus, indeed a group of genesleading to aberrant mesenchymal growth including but not restricted toHMGI-C and HMGI(Y) can be defined.

[0116] 2. Material and methods

[0117] 2.1. Chromosome Preparation

[0118] Chromosome preparation followed routine methods. Cells weretreated with 30 μl colcemide (10 μg/ml) for 2-3 h and then harvestedusing the trypsin method (0.05% trypsin, 0.02% EDTA) followed by ahypotonic shock in six fold diluted medium TC 199 for 20 minutes at roomtemperature and methanol:acetic acid (3:1) fixation. Chromosomes werethen GTG-banded.

[0119] 2.2. In Situ Hybridization

[0120] In situ hybridisation was performed as outlined in one of theprevious examples.

[0121] 2.3. PAC Library Screening

[0122] The PAC library (Genome Systems Library Screening Service, St.Louis, Mo., USA) was screened by PCR with a primer set specific for theHMGI(Y) gene. For screening we designed the forward primer with thesequence:

[0123] 5′-CTC CAA GAC AGG CCT CTG ATG T-3′ (intron 3) and the reverseprimer:

[0124] 5′-ACC ACA GGT CCC CTT CAA ACT A-3′ (intron 3) giving rise to afragment of 338 bp. For amplification the following thermal cycling wasused: 94° C., 5 min, (94° C., 1 min, 59° C., 1 min, 72° C., 2 min)×30,72° C., 10 min.

[0125] 2.4. DNA Preparations From PAC Clones

[0126] Bacterial colonies containing single PAC clones were inoculatedinto LB medium and grown overnight at 37° C. 660 μl of the overnightculture were diluted into 25 ml of LB medium and grown to an OD₅₅₀ of0.05-0.1. By addition of IPTG to a final concentration of 0.5 mM the P1lytic replicon was induced. After addition of IPTG, growth was continuedto an OD₅₅₀ of 0.5-1.5, and plasmid DNA was extracted using the alkalinelysis procedure recommended by Genome Systems.

[0127] 3. Results

[0128] The primer set for screening the human PAC library was designedfrom sequences belonging to intron 3 of HMGI(Y). Because of sequencehomology between HMGI-C and HMGI(Y) the amplified sequence of 338 bp wastested by homology search to be specific exclusively for HMGI(Y).Library screening resulted in three positive PAC clones that had anaverage insert length of approximately 100 kb. Two of these clones(Pac604, Pac605) were used for the following FISH studies. In order toprove if HMGI(Y) is rearranged in tumors with translocations involving6p21.3 in either simple or complex form we performed FISH analysis onmetaphase spreads from four primary pulmonary chondroid hamartomas andtwo endometrial polyps all with 6p21.3 abnormalities. For each case 20metaphases were scored. At least one of the two PAC clones Pac604 andPac605 described above was across the breakpoint in all six casesanalyzed. These results clearly show that the breakpoints of the tumorswith 6p21 aberrations investigated herein are clustered either within heHMGI(Y) gene or its close vicinity.

Example 4

[0129] Hybrid HMGI-C in Lipoma Cells.

[0130] cDNA clones of the chromosome 3-derived lipoma-preferred partnergene LPP (>50 kb) were isolated and the nucleotide sequence thereofestablished. Data of a composite cDNA are shown in FIG. 4. An openreading frame for a protein (612 amino acids (aa)) with amino acidsequence similarity (over 50%) to zyxin of chicken was identified. Zyxinis a member of the LIM protein family, whose members all possessso-called LIM domains [78]. LIM domains are cysteine-rich, zinc-bindingprotein sequences that are found in a growing number of proteins withdivers functions, including transcription regulators, proto-oncogeneproducts, and adhesion plaque constituents. Many of the LIM familymembers have been postulated to play a role in cell signalling andcontrol of cell fate during development. Recently, it was demonstratedthat LIM domains are modular protein-binding interfaces [79]. Likezyxin, which is present at sites of cell adhesion to the extracellularmatrix and to other cells, the deduced LPP-encoded protein (FIG. 6)possesses three LIM domains and lacks classical DNA-bindinghomeodomains.

[0131] In 3′-RACE analysis of Li-501/SV40, a HMGl-C containing fusiontranscript was identified from which a hybrid protein (324 aa) could bepredicted and which was subsequently predicted to consist of the threeDBDs (83 aa) of HMGl-C and, carboxy-terminally of these, the three LIMdomains (241 aa) encoded by LPP. In PCR analysis using approriate nestedamplimer sets similar HMGI-C/LPP hybrid transcripts were detected invarious primary lipomas and lipoma cell lines carrying a t(3;12) andalso in a cytogenetically normal lipoma. These data reveal that thecytogenetically detectable and also the hidden t(3;12) translocations inlipomas seem to result consistently in the in-phase fusion of theDNA-binding molecules of HMGl-C to the presumptive modularprotein-binding interfaces of the LPP-encoded protein, thereby replacingthe acidic domain of HMGl-C by LIM domains. Consequently, theseprotein-binding interfaces are most likely presented in the nuclearenvironment of these lipoma cells, where they might affect geneexpression, possibly leading to aberrant growth control. Out of thelarge variety of benign mesenchymal tumors with chromosome 12q13-q15aberrations, this is the first example of a chromosome translocationpartner contributing recurrently and consistently to the formation of awell-defined tumor-associated HMGl-C fusion protein.

[0132]FIG. 5 shows the cDNA sequence of the complete isolated LPP gene.

Example 5

[0133] Diagnostic Test for Lipoma

[0134] A biopsy of a patient having a lipoma was taken. From thematerial thus obtained total RNA was extracted using the standardTRIZOL™ LS protocol from GIBCO/BRL as described in the manual of themanufacturer. This total RNA was used to prepare the first strand ofcDNA using reverse transcriptase (GIBCO/BRL) and an oligo dT(17) primercontaining an attached short additional nucleotide stretch. The sequenceof the primer used is as described in Example 2, under point 2.5. RNaseH was subsequently used to remove the RNA from the synthesized DNA/RNAhybrid molecule. PCR was performed using a gene specific primer (Example2, point 2.5.) and a primer complementary to the attached shortadditional nucleotide stretch. The thus obtained PCR product wasanalysed by gel electroforesis. Fusion constructs were detected bycomparing them with the background bands of normal cells of the sameindividual.

[0135] In an additional experiment a second round of hemi-nested PCR wasperformed using one internal primer and the primer complementary to theshort nucleotide stretch. The sensitivity of the test was thussignificantly improved.

[0136]FIG. 8 shows a typical gel.

Example 6

[0137] Aberrations of 12q14-15 and 6p21 in Pulmonary ChondroidHamartomas

[0138] 1. Introduction

[0139] Pulmonary chondroid hamartomas (PCH) are often detected duringX-ray examination of the lung as so-called coin lesions. However, lungmetastases of malignant tumors and rarely lung cancers can also presentas coin lesions. This example shows that FISH requiring a minimal amountof tumor cells can be used to correctly distinguish between the majorityof PCHs and malignant tumors. Thus the test can successfully be appliede.g. to tumor cells obtained by fine needle aspiration.

[0140] 2. Materials and Methods

[0141] Samples from a total of 80 histologically characterized PCHs wereincluded in this study. Cell cultures, chromosome preparations and FISHwere obtained or performed as described in the previous examples.

[0142] 3. Results

[0143] Cytogenetic studies revealed that of the 80 PCHs studiedcytogenetically 51 revealed detectable aberrations involving either12q14-15 or 6p21. By FISH using either a pool of cosmids belonging tothe HMGI-C gene or using the PAC clones of HMGI(Y) described in theprevious example we were able to detect hidden structural rearrangementsof those regions in 4 additional cases (3 with 12q and one with 6 pinvolvement). Therefore, the FISH test alone can be used for a kit toprecisely detect the rearrangement of either the HMGI-C or the HMGI(Y)gene rearrangements in more than 50% of the PCHs and is thus a valuableadditonal tool for the diagnosis of these tumors (without beingrestricted to this type of tumors as shown in two of the otherexamples).

Example 7

[0144] Diagnosis of Soft Tissue Tumors Particularly of Adipocytic Origin

[0145] 1. Introduction

[0146] Adipocyte tissue tumors often cause diagnostic difficultiesparticularly when material taken from fine needle aspiration biopsies orcryosections has to be evaluated. This examples demonstrates thevalidity of the FISH test for the differential diagnosis of adipocytetissue tumors and rare soft tissue tumors.

[0147] 2. Materials and Methods

[0148] 2.1. Tumor Samples

[0149] Tumor samples from three soft tissue tumors were investigated byFISH. Sample one (1) was from a adipocytic tumor and histologically itwas either an atypical lipoma or a well-differentiated liposarcoma. Thesecond case (tumor 2) was diagnosed to be most likely a myxoidliposarcoma but other types of malignant soft tissue tumors includingaggressive angiomyxoma were also considered. The third tumor (tumor 3)was also of adipocytic origin and both a lipoma and a welldifferentiated liposarcoma were considered.

[0150] 2.2. Isolation of Cells and FISH

[0151] The tumor samples were enzymatically disaggregated followingroutine methods. The resulting single cell suspensions were centrifugedand the suspensions were fixed using methanol:glacial acetic acid (3:1)at room temperature for 1 hour. The cell suspensions were then droppedon clean dry slides and allowed to age for 6 hours at 60° C. FISH wasperformed using molecular probes from the HMGI-C gene as described inthe previous examples.

[0152] 3. Results

[0153] At the interphase level tumor 1 and 2 both showed split signalsfor one of the alleles. These findings are compatible with the diagnosisof benign tumors i.e. an atypical lipoma in the first case and anaggressive angiomyxoma in the second case. They allowed to rule out thepresence of malignant adipocytic tissue tumors.

[0154] In the third case the FISH revealed a high degree ofamplification of the MAR region or part of it. Since the amplificationunits observed in giant marker or ring chromosomes inwell-differentiated liposarcomas can involve the MAR region thesefindings leads to the diagnosis of a well-differentiated liposarcoma.The three cases presented within this example show the usefulness of theDNA probes described. They can be used in a kit for a relatively simpleand fast interphase FISH experiment offering an additional tool for thediagnosis of soft tissue tumors.

Example 8

[0155] Expression of the HMGI-C Gene in Normal Tissue

[0156] 1. Introduction

[0157] It is the aim of this example to show that the expression of theHMGI-C gene is mainly restricted to human tissues during embryonic andfetal development. In contrast, in most normal tissues of the adultparticularly, including those tissues and organs tumors with HMGI-Crearrangements can arise from, no expression can be noted. Thisindicates that even the transcriptional re-activation of the gene caninititate tumorigenesis. On the other hand it underlines the usefulnessof antisense strategies (including those antisense molecules directedtowards the normal HMGI-C mRNA) to inhibit or stop tumor growth.

[0158] 2. Materials and Methods

[0159] 2.1. Tissue Samples

[0160] All adult tissue samples used for this study were taken fromsurgically removed tissue frozen in liquid nitrogen within a period of15 min after removal. Most of the samples were from adjacent normaltissue removed during tumor surgery. In detail we have used 8 samplestaken from fat tissues at various anatomical sites, 20 samples takenfrom myometrial tissue, 8 samples taken from lung tissue, 4 samplestaken from the salivary glands (Glandula parotis and Glandulasubmandibularis), one tissue sample taken from the heart muscle, 25samples taken from breast tissue from patients of different ages, 2samples from the brain, 3 liver samples, 7 samples taken from renaltissue, and embryonic/fetal tissue (extremeties, 6 samples) fromembryos/fetuses (10-14th gestational week) after abortion fromsocio-economic reasons.

[0161] In addition, three cell lines were used: As a control for HMGI-Cexpression we used the hepatoma cell line Hep 3B and the cell line L14established from a lipoma with the typical translocation t(3;12). HeLacells were used as a negative control because RT experiments reproducedfor 10 times did not reveal HMGI-C expression in our own studies.

[0162] 2.2. RT-PCR for the Expression of HMGI-C

[0163] 100 mg of tissue sample was homogenized, and RNA was isolatedusing the trizol reagent (GibcoBRL, Eggenstein, Germany) containingphenol and isothiocyanate. cDNA was synthesized using apoly(A)-oligo(dt)17 primer and M-MLV reverse transcriptase (GibcoBRL,Eggenstein, Germany). Then, a hemi-nested PCR was performed.

[0164] For first and second PCR the same lower primer (Revex 4) (5′-TCCTCC TGA GCA GGC TTC-3′ (exon 4/5)) was used. In the first round of PCRthe specific upper primer (SE1) (5′-CTT CAG CCC AGG GAC AAC-3′ (exon1)), and in the second round of PCR the nested upper primer (P1) (5′-CGCCTC AGA AGA GAG GAC-3′ (exon 1)) was used. Both rounds of PCR wereperformed in a 100 μl volume containing 10 mM Tris/HCl pH 8.0, 50 mMKCl, 1.5 mM MgCl₂, 0.001 % gelatin, 100 μM dATP, 100 μM dTTP, 100 μMdGTP, 100 μM dCTP, 200 nM upper primer, 200 nM lower primer, and 1unit/100 μl AmpliTaq polymerase (Perkin Elmer, Weiterstadt, Germany).Amplification was performed for 30 cycles (1 min 94° C., 1 min 53° C., 2min 72° C.). As template in the first round of PCR cDNA derived from 250ng total RNA, and in the second round of PCR 1 μl of the first PCRreaction mix was used.

[0165] 2.3. Control Assay for Intact mRNA/cDNA

[0166] As control reaction for intact RNA and cDNA PCR a test based onthe amplification of, the cDNA of the housekeeping gene glyceraldehyde3-phosphate dehydrogenase (GAPDH). PCR reaction was performed for 35cycles under the same conditions as described above for the first roundof PCR of HMGI-C expression.

[0167] 3. Results

[0168] As for the expression studies all experiments were repeated atleast twice. To assure that all RNA and cDNA preparations used for theRT-PCRs were intact (otherwise resulting in false negative results)routinely a RT-PCR for expression of the housekeeping gene GAPDH wasperformed. A positive GAPDH RT-PCR results in a 299 bp fragment. Onlysamples revealing a positive GAPDH RT-PCR were included in this study.As the result of RT-PCR in HMGI-C positive cells such as Hep 3B and L14a specific 220 bp fragment is detectable. HeLa cells did not show anexpression of HMGI-C. Except for two myometrial samples (most likely dueto myomas at a submicroscopic level) all normal tissue samples takenfrom adult individuals did not show any detectable level of HMGI-Cexpression. In contrast, all fetal/embryonic tissues tested revealedHMGI-C expression.

Example 9

[0169] Expression of the HMGI-C Gene as a Diagnostic Tool for the EarlyDetection of Leukemias

[0170] 1. Introduction

[0171] Cytogenetically detectable aberrations affecting the HMGI-C genehave been found in a variety of benign solid tumors of mesenchymalorigin. Apparently, the aberrations also lead to the transcriptionalactivation of the gene. Since blood cells are also of mesenchymalorigin, it was tempting to check leukemic cells for HMGI-C expression.The present example shows that the activation of the gene in cells ofthe peripheral blood is a suitable marker indicating immaturecells/abnormal stem cells found in leukemias. Since the expression ofHMGI-C can be determined with a high degree of sensitivity the RT-PCRfor the expression of the gene can be used for a very early detection ofvarious hematological diseases.

[0172] 2. Materials and Methods

[0173] Samples from peripheral blood of 27 patients with different typesof leukemias including 19 patients with Philadelphia-chromosome positiveCML, 5 patients with AML, and 3 patients with ALL were used fordetermination of HMGI-C expression. Blood samples from 15 healthyprobands served as controls.

[0174] RT-PCR for the expression of HMGI-C was performed as outlined inexample 8.

[0175] 3. Results

[0176] Whereas expression of HMGI-C was clearly detectable in all bloodsamples from leukemic patients there was no expression noted in any ofthe blood samples taken from the control persons. There is no evidencethat the transcriptional activation of the gene is due to mutationsaffecting the gene or its surroundings. It is more reasonable to assumethat the activation is rather a secondary effect related to theimmaturity of the cells or their abnormal proliferation. However, thehigh and even improvable sensitivity makes e.g. a kit based on theRT-PCR for the expression of the HMGI-C gene a very suitable diagnostictool.

Example 10

[0177] The Transcriptional Re-Expression of the HMGI-C Gene can Lead tothe Initiation of the Tumors

[0178] 1. Introduction

[0179] This example clearly shows that for some tumor entitieschromosomal breakpoints located 5′ of the HMGI-C gene do also existindicating that the transcriptional up-regulation of the gene issufficient to initiate growth of the corresponding tumor types.

[0180] 2. Materials and Methods

[0181] 2.1. Cell Culture

[0182] After surgery the tumor samples (three pulmonary chondroidhamartomas, one uterine leiomyoma) were washed with Hank's solutionsupplemented with penicillin (200 IU/ml) and streptomycin (200 μg/ml).Tumors were disaggregated with collagenase for 5-6 h at 37° C. Thesuspension containing small fragments and single cells was resuspendedin culture medium TC 199 with Earle's salts supplemented with 20% fetalbovine serum, 200 IU/ml penicillin, and 200 μg/ml streptomycin.

[0183] 2.2. Chromosome Preparations

[0184] Chromosome preparation followed routine methods. Cells weretreated with 30 μl colcemide (10 μg/ml) for 2-3 h and then harvestedusing the trypsin method (0.05% trypsin, 0.02% EDTA) followed by ahypotonic shock in six fold diluted medium TC 199 for 20 minutes at roomtemperature and methanol:acetic acid (3:1) fixation. Chromosomes werethen GTG-banded.

[0185] 2.3. FISH Studies

[0186] To identify the chromosomes unambiguously, FISH was performedafter GTG-banding of the same metaphase spreads. As DNA probes we usedfive cosmids belonging to a YAC-contig overspanning the HMGI-C gene asdescribed in the Legenda of FIG. 2. Three of these cosmids (27E12,185H2, 142H1) are mapping to the third intron of HMGI-C, whereas cosmids260C7 and 245E8 are localized at the 3′ or the 5′ end respectively. Theslides were analyzed using a Zeiss (Zeiss, Oberkochem, Germany) Axioplanfluorescence microscope. Results were processed and recorded with thePower Gene Karyotyping System (PSI, Halladale, Great Britain). Rapidamplification of cDNA ends (RACE) was performed as described in one ofthe former examples.

[0187] 3. Results

[0188] All four tumors showed the same type of cytogenetic abnormality,i.e. the presence of 47 chromosomes including two apparently normalchromosomes 12 and an additional derivative 14 der(14)t(12;14)(q14-15;q24) but without a corresponding der(12). Since the 3′-5′orientation of the HMGI-C is towards the centromere a single breakwithin the HMGI-C gene would have led to the loss of its 5′ part alongwith the loss of the der(12). We have therefore performed a series ofFISH experiments in order to determine the breakpoints more precisely.Using the five cosmids 260C7, 27E12, 185H2, 142H1, and 245E8hybridization signals of the same intensity were observed at both normalchromosomes 12 and at the additional der(14). The FISH results revealedthat in all four cases chromosomal breakpoints were located 5′ of theHMGI-C gene.

[0189] The breakpoint assignment in all four cases 5′ of the HMGI-C genefits well with the results of the RACE-PCR. In addition to the normalHMGI-C transcripts we were able to detect aberrant transcripts in allthree tumors. Sequences showed that they were not derived fromchromosome 14 but from intron 3 of HMGI-C probably due to cryptic splicesites. However, the RACE results revealed that there was indeed HMGI-Cexpression in all four cases.

Example 11 Re-Differentiation of Leukemic Cells

[0190] 1. Introduction

[0191] Expression of the HMGI-C gene is frequently strongly elevated ina wide variety of tumors, solid tumors as well as leukemias. It wasspeculated that the HMGI-C protein might play a key role intransformation of cells. This example shows that expression of theHMGI-C gene can be strongly reduced by expressing antisense HMGI-Csequences and that reduction of HMGI-C levels in tumor cells results inreversion of the transformed phenotype. Thus the expression oradministration of antisense molecules can be successfully appliedtherapeutically.

[0192] 2. Materials and Methods

[0193] 2.1. Tumor Cell Lines

[0194] Tumor cell lines were generated from a primary malignant salivarygland tumor and a primary breast carcinoma. Cell lines were establishedas described by Kazmierczak, B., Thode, B., Bartnitzke, S., Bullerdiek,J. and Schloot, W., “Pleomorphic adenoma cells vary in theirsusceptibility to SV40 transformation depending on the initialkarytotype.”, Genes Chrom. Cancer 5:35-39 (1992).

[0195] 2.2. Assay of the Transformed State

[0196] Soft agar colony assays were performed as described by Macphersonand Montagnier, “Agar suspension culture for the selective assays ofcells transformed by polyoma virus.” Virology 23, 291-294 (1964).

[0197] Salivary gland and breast tumor cells were propagated in TC199culture medium with Earle's salts, supplemented with 20% fetal bovineserum (GIBCO), 200 IU/ml penicillin, and 200 μg/ml streptomycin.

[0198] Tumorigenicity of the transfected salivary gland (AD64) andbreast cell lines was tested by injecting cells subcutaneously intoathymic mice.

[0199] 2.3. Transfection Assay

[0200] Transfections were performed using various protocols, namely:

[0201] 1. The calcium phosphate procedure of Graham and Van der Eb (“Anew technique for the assay of the infectivity of human adenovirus.”Virology 52, 456-467 (1973) ).

[0202] 2. Lipofection: Transfections were carried out usingliposome-mediated DNA transfer (lipofectamine, GibcoBRL) according tothe guidelines of the manufacturer.

[0203] 2.4. Antisense Constructs

[0204] Sense and antisense constructs of the HMGI-C gene were obtainedby inserting human HMGI-C cDNA sequences in both the sense and antisenseorientation in expression vectors under the transcriptional control ofvarious promoter contexts, e.g. the long terminal repeat of Moloneymurine leukemia virus, a CMV promoter, or the early promoter of SV40.For example, the CMV/HMGI-C plasmid was constructed by cloning a humanHMGI-C cDNA fragment containing all coding sequences of human HMGI-C inpRC/CMV (Invitogen) allowing expression under control of the humancytomegalovirus early promoter and enhancer, and selection for G418resistance.

[0205] 3. Results

[0206] 3.1. Reversion of the Transformed Phenotype

[0207] Reversion of the transformed phenotype was observed in breast andsalivary gland tumors cells after induction of antisense HMGI-Cexpression in these tumor cells. A strong reduction in tumorigenicitywas observed as measured by soft agar colony assay and in vivo inathymic mice. Immunoprecipitation and Western blot analysis indicated astrong reduction of HMGI-C protein levels in the cells expressingantisense HMGI-C sequences. Therefore, this approach can be usedtherapeutically in tumors with involvement of HMGI-C.

Example 12

[0208] Animal Tumor Models Involving HMGI-C as Tools in In VivoTherapeutic Drug Testing.

[0209] On the basis of the acquired HMGI-C knowledge, animal tumormodels can be developed as tools for in vivo drug testing. To achievethis objective (for instance for uterine leiomyoma), two approaches canbe used, namely gene transfer (generation of transgenic animals) on theone hand and gene targeting technology (mimicking in vivo of a specificgenetic aberration via homologous recombination in embryonic stem cells(ES cells)) on the other.

[0210] These technologies allow manipulation of the genetic constitutionof complex living systems in specific and pre-designed ways. Forextensive technical details, see B. Hogan, R. Beddington, F.Constantini, and E. Lacy; In: Manipulating the mouse embryo, ALaboratory Manual. Cold Spring Harbor Press, 1994; ISBN 0-87969-384-3.

[0211] To aim at the inactivation or mutation of the HMGI-C gene,specifically in selected cell types and selected moments in time, therecently described Cre/LoxP system can be used (Gu, H. et al. Deletionof a DNA polymerase β gene segment in T cells using cell type-specificgene targeting. Science 265, 103-106, 1994). The Cre enzyme is arecombinase from bacteriophage P1 whose physiological role is toseparate phage genomes that become joined to one another duringinfection. To achieve so, Cre lines up short sequences of phage DNA,called loxP sites and removes the DNA between them, leaving one loxPsite behind. This system has now been shown to be effective in mammaliancells in excising at high efficiency chromosomal DNA. Tissue-specificinactivation or mutation of a gene using this system can be obtained viatissue-specific expression of the Cre enzyme.

[0212] As an example, the development of animal model systems foruterine leiomyoma using a member of the MAG gene family will be outlinedbelow, such that the models will be instrumental in in vivo testing oftherapeutic drugs.

[0213] Two approaches may be followed:

[0214] a) in vivo induction of specific genetic aberrations as observedin human patients ((conditional) gene (isogenic) targeting approach);and

[0215] b) introduction of DNA constructs representative for the geneticaberrations observed in patients (gene transfer approach).

[0216] DNA constructs to be used in gene transfer may be generated onthe basis of observations made in patients suffering from uterineleiomyoma as far as structure and expression control are concerned; e.g.HMGI-C fusion genes with various translocation partner genes, especiallythe preferential translocation partner gene of chromosome 14 located inthe YAC contig represented by CEPH YACs 6C3, 89C5, 308H7, 336H12, 460A6,489F4, 902F10, 952F5, 958C2, 961E1, and 971F5, truncated genes encodingbasically the three DNA binding domains of HMGI-C, and complete HMGI-Cor derivatives of HMGI-C under control of a strong promoter.

Example 13

[0217] The Preparation of Antibodies Against HMGI-C

[0218] One type of suitable molecules for use in diagnosis and therapyare antibodies directed against the MAG genes. For the preparation ofrabbit polyclonal antibodies against HMGI-C use was made of thefollowing three commercially available peptides:

[0219] (H-ARGEGAGQPSTSAQGQPAAPAPQIR) 8-Multiple Antigen Peptide

[0220] (H-SPSKAAQKKAEATGEKR) 8-MAP

[0221] (H-PRKWPQQVVQKKPAQEE) 8-MAP

[0222] obtainable from Research Genetics Inc., Huntsville, Ala., USA.The polyclonal antibodies were made according to standard techniques.TABLE 1 ANALYSIS OF YAC CLONES CEPH- Size Landmark Landmark Code (kb)left # right # Chimeric 183F3 715 [RM10] YES (L + R) 70E1 450 RM29U27125 ND 95F1 390 RM30 U29054 ND 201H7 320 RM13 U29051 RM14 U29053 ND186G12 320 ND 354B6 280 YES (R) 126G8 410 ND 258F11 415 RM4 U29052 ND320F6 290 RM5 U29050 RM21 U29047 ND 234G11 475 RM7 U29046 ND 375H5 290ND 262E10 510 [RM15] RM16 U29048 YES (L) 181C8 470 RM26 U29045 ND 107D1345 RM31 U29043 ND 499C5 320 RM44 U29044 RM45 U29037 ND 340B6 285 ND532C12 400 RM45 U29041 ND 138C5 510 [RM59] RM65 U29042 YES (L) 145F2 490RM60 U29030 RM66 U29040 ND 105E8 340 RM57 U29033 RM63 U29038 ND 55G1 365RM56 U29031 RM62 U29039 ND 103G7 370 RM85 U29025 RM80 U29036 ND 295B10295 RM77 U29035 RM81 U29026 ND 338C2 200 RM78 U29034 RM82 U29029 ND391C12 160 [RM79] RM83 U29027 YES (L) 476A11 225 [RM87] RM84 U29032 YES(L) 138F3 460 RM90 U29028 RM54 U29015 ND 226E7 500 RM48 U29024 RM54U29015 ND 499E9 375 RM51 U29016 YES (R) 312F10 580 [RM50 ] RM69 U29021YES (L) 825G7 950 ND 34B5 315 RM88 U29020 RM89 U29013 ND 94A7 610 YES(R) 305B2 660 YES (L) 379H1 280 RM104 U29014 RM105 U29309 ND 444E6 350RM92 U29017 RM93 U29310 ND 446H3 370 RM94 U29011 RM95 U29018 ND 403B12380 ND 261E5 500 RM102 U29012 RM103 U26689 ND 78B11 425 ND 921B9 1670 ND939H2 1750 ND 188H7 360 ND 142F4 390 ND 404E12 350 ND 164A3 375 ND244B12 415 RM105 U29007 RM107 U29008 ND 275H4 345 RM108 U29004 RM109U29005 ND 320F9 370 ND 51P8 450 ND 242A2 160 CH1 U29006 ND 253H1 400 ND303F11 320 ND 322C8 410 CH2 U29303 ND 208G12 370 RM96 U29002 RM97 U27135ND 341C1 270 RM98 U26647 RM99 U27130 ND 354F1 270 ND 452E1 270 CH5U27136 ND 41A2 310 ND 934D2 1370 ND 944E8 1290 CH8 U25792 ND 2G11 350 ND755D7 1390 YES (L) 365A12 370 ND 803C2 1080 ND 210C1 395 RM70 U28998RM86 U27133 ND 433C8 350 RM73 U29000 RM76 U27132 ND 402A7 500 RM41U28994 [RM42] YES (R) 227E8 465 RM53 U27134 RM55 U23996 ND 329F9 275RM72 U28793 RM75 U23997 ND 261ES 395 [RM71] RM74 U23995 YES (L) 348F2370 [RM136] YES (R) 6F3 320 RM35 U27140 RM36 U27141 59F12 430 RM34U28794 RM33 U27131 265H3 300 RM40 U25999

[0223] TABLE II PCR Primers STS name (STS 12-) Nucleotide sequence 5′-3′Product size (bp) T_(orr) (° C.) CH1 TGGGACTAACGGATTTTCAA 213 58TGTGGTTCATTCATGCATTA CH2 TCCATCATCATCTCAAAACA 145 58CTCTACCAAATGGAATAAACAG CH5 GCAGCTCAGGCTCCTTCCCA 143 58TGGCTTCCTGAAACGCGAGA CH8 TCTCCACTGCTTCCATTCAC 147 58ACACAAAACCACTGGGGTCT CH9 CAGCTTTGGAATCAGTGAGG 262 58CCTGGGGAAGAGGAGTAAAG RM1 GAGCTTCCATATCTCATCC 308 60 ATGCTTGTGTGTGAGTGGRM4 TTTGCTAAGCTAGGTGCC 236 60 AGCTTCAAGACCCATGAG RM5 CAGTTCTGAGACTGCTTG324 60 TAATAGCAGGGACTCAGC RM7 CTTGTCTCATTCTTTTAAAGGG 538 58CACCCCTTTTTAGATCCTAC RM13 GAATGTTCATCACAGTGCTG ±500 58AATGTGAGGTTCTGCTGAAG RM14 TTCTCATGGGGTAAGGACAG 158 58AAAGCTGCTTATATAGGGAATC RM16 CCTTGGCTTAGATATGATACAC 252 58GCTCTTCAGAAATATCCTATGG RM21 CCTTAGCAGTTGCTTGTCTG 290 58TCGTCACAGGACATAGTCAC RM26 TCTATGGTATGTTATACAAGATG 102 58CAGTGAGATCCTGTCTCTA RM31 TCTGTGATGTTTTAAGCCACTTAG 239 56AATTCTGTGTCCCTGCCACC RM33 ATTCTTCCTCACCTCCCACC ±600 60AATCTGCAGAGAGGTCCAGC RM34 AATTCTCCATCTGGGCCTGG ±600 60GAACGCTAAGCATGTGGGAG RM36 CTCCAACCATGGTCCAAAAC 296 60GACCTCCAGTGGCTCTTTAG RM46 ACCATCAGATCTGGCACTGA 241 57TTACATTGGAGCTGTCATGC RM48 TCCAGGACATCCTGAAAATG 391 58AGTATCCTGCACTTCTGCAG RM51 GATGAACTCTGAGGTGCCTTC 311 60TCAAACCCAGCTTTGACTCC RM53 GTCTTCAAAACGCTTTCCTG 333 60TGGTTTGCATAATGGTGATG RM60 TACACTACTCTGCAGCACAC 94 58TCTGAGTCAATCACATGTCC RM69 CTCCCCAGATGATCTCTTTC 236 58CGGTAGGAAATAAAGGAGAG RM72 TATTTACTAGCTGGCCTTGG 101 62CATCTCAGGCACACACAATG RM76 ATTCAGAGAAGTGGCCAAGT 496 58GGGATAGGTCTTCTGCAATC RM85 TCCAACAATACTGAGTGACC 435 58TCCATTTCACTGTAGCACTG RM86 GTAATCAACCATTCCCCTGA 203 56AAAATAGCTGGTATGGTGGC RM90 ACTGCTCTAGTTTTCAAGGA 257 58AATTTACCTGACAGTTTCCT RM93 GCATTTGACGTCCAATATTG 347 60ATTCCATTGGCTAACACAAG RM98 GCAAAACTTTGACTGAAACG 356 58CACAGAGTATCGCACTGCAT RM99 AAGAGATTTCCCATGTTGTG 240 58CTAGTGCCTTCACAAGAACC RM103 AATTCTTGAGGGGTTCACTG 199 60TCCACACTGAGAGCTTTTCA RM110 GCTCTACCAGGCATACAGTG 439 60ATTCCTAGCATCTTTTCACG RM111 ATATGCATTAGGCTCAACCC 328 58ATCCCACAGGTCAACATGAC RM130 ATCCTTACATTTCCAGTGGCATTCA 312 58CCCAGAAGACCCACATTCCTCAT RM131 TTTTAAGTTTCTCCAGGGAGGAGAC 336 58AATAGGCTCTTTGGAAAGCTGGAGT RM132 TCTCAGCTTAATCCAAGAAGGACTTC 376 58GGCATATTCCTCAACAATTTATGCTT RM133 TGGAGAAGCTATGGTGCTTCCTATG 225 58TGACAAATAGGTGAGGGAAAGTTGTTAT ESTRO1096 TCACACGCTGAATCAATCTT 188 58CAGCAGCTGATACAAGCTTT IFNG TGTTTTCTTTCCCGATAGGT 150 52CTGGGATGCTCTTCGACCTC Rap1B CCATCCAACATCTTAAATGGAC 149 58CAGCTGCAAACTCTAGGACTATT

[0224] TABLE 3 Genome Data Base accession numbers (D-numbers) of thevarious sequences indicated in Figure 1. Genome Data Base (41 rowsaffected) pcr locus_symbol pcr pcr_gdb_id locus locus_gdb_idCH1-lower/CH1-upper D12S1484 CH1-lower/CH1-upper G00-595-292 D12S1484G00-595-415 CH2-lower/CH2-upper D12S1485 CH2-lower/CH2-upper G00-595-295D12S1485 G00-595-416 CH5-lower/CH5-upper D12S1486 CH5-lower/CH5-upperG00-595-298 D12S1486 G00-595-417 CH8-lower/CH8-upper D12S1487CH8-lower/CH8-upper G00-595-301 D12S1487 G00-595-418 CH9-lower/CH9-upperD12S1488 CH9-lower/CH9-upper G00-595-304 D12S1488 G00-595-419CH2-lower/CH2-upper D12S1489 CH2-lower/CH2-upper G00-595-307 D12S1489G00-595-420 CH3-lower/CH3-upper D12S1490 CH3-lower/CH3-upper G00-595-310D12S1490 G00-595-421 CH4-lower/CH4-upper D12S1491 CH4-lower/CH4-upperG00-595-313 D12S1491 G00-595-422 RM13-lower/RM13-upper D12S1492RM13-lower/RM13-upper G00-595-316 D12S1492 G00-595-423RM14-lower/RM14-upper D12S1493 RM14-lower/RM14-upper G00-595-319D12S1493 G00-595-424 RM16-lower/RM16-upper D12S1494RM16-lower/RM16-upper G00-595-322 D12S1494 G00-595-425RM25-lower/RM25-upper D12S1507 RM26-lower/RM26-upper G00-595-325D12S1495 G00-595-426 RM26-lower/RM26-upper D12S1495RM-29-lower/RM29-upper G00-595-328 D12S1496 G00-595-427RM31-lower/RM31-upper D12S1497 RM31-lower/RM31-upper G00-595-331D12S1497 G00-595-428 RM33-lower/RM33-upper D12S1498RM33-lower/RM33-upper G00-595-334 D12S1498 G00-595-429RM34-lower/RM34-upper D12S1499 RM34-lower/RM34-upper G00-595-337D12S1499 G00-595-430 RM36-lower/RM36-upper D12S1500RM36-lower/RM36-upper G00-595-340 D12S1500 G00-595-431RM46-lower/RM46-upper D12S1501 RM46-lower/RM46-upper G00-595-343D12S1501 G00-595-432 RM48-lower/RM48-upper D12S1502RM48-lower/RM48-upper G00-595-346 D12S1502 G00-595-433RM51-lower/RM51-upper D12S1503 RM51-lower/RM51-upper G00-595-349D12S1503 G00-595-434 RM53-lower/RM53-upper D12S1504RM53-lower/RM53-upper G00-595-352 D12S1504 G00-595-435RM60-lower/RM60-upper D12S1505 RM60-lower/RM60-upper G00-595-355D12S1505 G00-595-436 RM69-lower/RM69-upper D12S1506RM69-lower/RM69-upper G00-595-358 D12S1506 G00-595-437RM72-lower/RM72-upper D12S1508 RM25-lower/RM25-upper G00-595-361D12S1507 G00-595-438 RM76-lower/RM76-upper D12S1509RM72-lower/RM72-upper G00-595-364 D12S1508 G00-595-439RM85-lower/RM85-upper D12S1510 RM76-lower/RM76-upper G00-595-367D12S1509 G00-595-440 RM86-lower/RM86-upper D12S1511RM85-lower/RM85-upper G00-595-370 D12S1510 G00-595-441RM90-lower/RM90-upper D12S1512 RM86-lower/RM86-upper G00-595-373D12S1511 G00-595-442 RM93-lower/RM93-upper D12S1513RM90-lower/RM90-upper G00-595-376 D12S1512 G00-595-443RM98-lower/RM98-upper D12S1514 RM93-lower/RM93-upper G00-595-379D12S1513 G00-595-444 RM99-lower/RM99-upper D12S1515RM98-lower/RM98-upper G00-595-382 D12S1514 G00-595-445RM-29-lower/RM29-upper D12S1496 RM99-lower/RM99-upper G00-595-385D12S1515 G00-595-446 RM103-lower/RM103-upper D12S1516RM103-lower/RM103-upper G00-595-388 D12S1516 G00-595-447RM108-lower/RM108-upper D12S1517 RM108-lower/RM108-upper G00-595-391D12S1517 G00-595-448 RM110-lower/RM110-upper D12S1518RM110-lower/RM110-upper G00-595-394 D12S1518 G00-595-449RM111-lower/RM111-upper D12S1519 RM111-lower/RM111-upper G00-595-397D12S1519 G00-595-450 RM121-lower/RM121-upper D12S1520RM121-lower/RM121-upper G00-595-400 D12S1520 G00-595-451RM130-lower/RM130-upper D12S1521 RM130-lower/RM130-upper G00-595-403D12S1521 G00-595-452 RM131-lower/RM131-upper D12S1522RM131-lower/RM131-upper G00-595-406 D12S1522 G00-595-453RM132-lower/RM132-upper D12S1523 RM132-lower/RM132-upper G00-595-409D12S1523 G00-595-454 RM133-lower/RM133-upper D12S1524RM133-lower/RM133-upper G00-595-412 D12S1524 G00-595-455

[0225] TABLE 4 FISH mapping of chromosome 12 breakpoints in primarybenign solid tumors to a subregion of MAR Fraction of tumors withbreakpoints within main Breakpoint breakpoint cluster Tumor type withinMAR region* Lipoma 6/6 6/6 Pleomorphic salivary 7/7 5/7 gland adenomaUterine leiomyoma 7/8 7/8 Hamartoma of the breast 1/1 1/1 Fibroadenomaof the breast 1/1 1/1 Hamartoma of the lung 8/9 8/9 Angiomyxoma 1/1 1/1

[0226] TABLE 5

LEGENDS TO THE FIGURES

[0227]FIG. 1

[0228] Long range physical map of a 6 Mb region on the long arm of humanchromosome 12 deduced from a YAC contig consisting of 75 overlappingCEPH YAC clones and spanning the chromosome 12q breakpoints as presentin a variety of benign solid tumors. The long range physical map of thecomposite genomic DNA covered by the YAC inserts is represented by ablack solid line with the relative positions of the various restrictionsites of rare cutting enzymes indicated. DNA regions in which additionalcutting sites of a particular restriction enzyme might be found areindicated by arrows. Polymorphic restriction endonuclease sites aremarked with asterisks. DNA markers isolated and defined by others aredepicted in green. DNA markers obtained by us are shown in boxes and arelabelled by an acronym (see also Table I and II). The relative positionsof these DNA markers in the long range physical map are indicated andthose corresponding to particular YAC ends are linked to these by adotted line. Some of the DNA markers have been assigned to a DNAinterval and this is indicated by arrows. For DNA markers in white boxesSTSs have been developed and primer sets are given in Table II. Forthose in yellow boxes, no primer sets were developed. The DNA intervalscontaining RAP1B, EST01096, or IFNG are indicated. Where applicable, Dnumber assignments are indicated. Below the long range physical map, thesizes and relative positions of the overlapping YAC clones fittingwithin the consensus long range restriction map are given as solid bluelines. DNA regions of YAC inserts not fitting within the consensus longrange restriction map are represented by dotted blue lines. CEPHmicrotiter plate addresses of the YAC clones are listed. The orientationof the YAC contig on chromosome 12 is given. The relative positions ofULCR12 and MAR are indicated by red solid lines labelled by thecorresponding acronyms. Accession numbers of STSs not listed in Table I:CH9 (#U27142) ; RM1 (#U29049); RM110 (#U29022); RM111 (#U29023); RM130(#U27139); RM131 (#U29001); RM132 (#U27138); RM133 (#U27137).Restriction sites: B: BssHII; K: KspI (=SacII); M: MluI; N: NotI; P:PvuI; Sf: SfiI.

[0229]FIG. 2

[0230] Contig of overlapping cosmids, long range restriction and STS mapspanning a segment of MAR of about 445 kb. Contig elements are numberedand defined in the list below. LL12NC01-derived cosmid clones are namedafter their microtiter plate addresses. GenBank accession numbers (#) ofthe various STSs are listed below. STSs are given in abbreviated form;e.g. RM33 instead of STS 12-RM33. A 40 kb gap between STSs “K” and “O”in the cosmid contig was covered by λ clones (clones 38 and 40) and PCRproducts (clones 37 and 39). The orientation of the contig on the longarm of chromosome 12 is given as well as the order of 37 STSs (indicatedin boxes or labelled with encircled capital letters). The slanted linesand arrows around some of the STS symbols at the top of the figure markthe region to which the particular STS has been assigned. It should benoted that the cosmid contig is not scaled; black squares indicate STSsof cosmid ends whereas the presence of STSs corresponding to internalcosmid sequences are represented by dots. Long range restriction map:Bs: BssHII; K: KspI (=SacII); M: MluI; N: NotI; P: PvuI; Sf: SfiI. Atthe bottom of the figure, detailed restriction maps are shown of thoseregions containing exons (boxes below) of the HMGI-C gene. Noncodingsequences are represented by open boxes and coding sequences by blackboxes. Estimated sizes (kb) of introns are as indicated. The relativepositions of the translation initiation (ATG) and stop (TAG) codons inthe HMGI-C gene as well as the putative poly-adenylation signal areindicated by arrows. Detailed restriction map: B: BamHI; E: EcoRI; H:HindIII. MAR: Multiple Aberration Region; DBD: DNA Binding Domain. 1 =140A3 11 = 142G8 21 = 124D8 31 = 59A1 41 = 128A2 51 = 65E6 2 = 202A1 12= 154A10 22 = 128A7 32 = 101D8 42 = 142H1 52 = 196E1 3 = 78F11 13 =163D1 23 = 129F9 33 = 175C7 43 = 204A10 53 = 215A8 4 = 80C9 14 = 42H7 24= 181C1 34 = 185H2 44 = 145E1 54 = 147G8 5 = 109B12 15 = 113A5 25 =238E1 35 = 189C2 45 = 245E8 55 = 211A9 6 = 148C12 16 = 191H5 26 = 69B136 = 154B12 46 = 154F9 56 = 22D8 7 = 14H6 17 = 248E4 27 = 260C7 37 =pRM150 47 = 62D8 57 = 116B7 8 = 51F8 18 = 33H7 28 = 156A4 38 = pRM144 48= 104A4 58 = 144D12 9 = 57C3 19 = 50D7 29 = 27E12 39 = PKXL 49 = 184A910 = 86A10 20 = 68B12 30 = 46G3 40 = pRM147 50 = 56C2 A = STS 12-EM12(#U27145) I = STS 12-CH12 (#U27153) Q = STS 12-RM120 (#U27161) B = STS12-EM30 (#U27146) J = STS 12-EM10 (#U27154) R = STS 12-RM118 (#U27162) C= STS 12-EM14 (#U27147) K = STS 12-EM37 (#U27155) S = STS 12-RM119(#U27163) D = STS 12-EM31 (#U27148) L = STS 12-RM146 (#U27156) T = STS12-EM2 (#U27164) E = STS 12-CH11 (#U27149) M = STS 12-RM145 (#U27157) U= STS 12-EM4 (#U27165) F = STS 12-EM18 (#U27150) N = STS 12-RM151(#U27158) V = STS 12-EM3 (#U27166) G = STS 12-EM11 (#U27151) O = STS12-EM16 (#U27159) W = STS 12-EM15 (#U27167) H = STS 12-CH10 (#U27152) P= STS 12-EM1 (#U27160) X = STS 12-EM17 (#U27168) STS 12-CH5 (#U27136)STS 12-CH9 (#U27142) STS 12-RM33 (#U27131) STS 12-RM53 (#U27134) STS12-RM76 (#U27132) STS 12-RM86 (#U27133) STS 12-RM98 (#U26647) STS12-RM99 (#U27130) STS 12-RH103 (#U26689) STS 12-RM130 (#U27139) STS12-RM132 (#U27138) STS 12-RM133 (#U27137) STS 12-RM151 (#U27158)

[0231]FIG. 3

[0232] Schematic representation of FISH mapping data obtained for tumorcell lines with chromosome 12q13-q15 aberrations, including 8 lipoma, 10uterine leiomyoma, and 8 pleomorphic salivary gland adenoma cell linesin consecutive experiments following our earlier FISH studies. Probesused included phage clones pRM144 (corresponding STSs: RM86 and RM130)and pRM147 (RM151), and cosmid clones 7D3 or 152F2 (RM103), 154F9 (CH9),27E12 (EM11), 211A9 (RM33), 245E8 (RM53), 185H2 (RM76), 202A1 (RM98),142H1 (RM99), 154B12 (RM132), and 124D8 (RM133). The DNA intervalbetween RM33 and RM98 is estimated to be about 445 kb. Dots indicateconclusive FISH experiments that were performed on metaphase chromosomesof a particular cell line using as molecular probe, a clone containingthe STS given in the box above. Solid lines indicate DNA intervals towhich a breakpoint of a particular cell line was concluded to bemapping. Open triangles indicate deletions observed during FISHanalysis. Open circles indicate results of FISH experiments on metaphasechromosomes of Li-501/SV40 cells with hybridization signals on acytogenetically normal chromosome 3. The positions of chromosome 12breakpoints of tumor cell lines mapping outside MAR are indicated byarrows. The molecularly cloned breakpoints of LM-30.1/SV40 andLM-608/SV40 are indicated by asterisks. Breakpoints in various uterineleiomyoma cell lines splitting cosmid 27E12 (EM11) are indicated by“across”.

[0233]FIG. 4

[0234] 3′-RACE product comprising the junction between part of theHMGI-C gene and part of the LPP gene. The primers used and the junctionare indicated. The cDNA synthesis was internally primed and not on thetrue poly(A) tail.

[0235]FIG. 5

[0236] Partial cDNA sequence of the LPP gene.

[0237]FIG. 6

[0238] Amino acid sequence of the LPP gene. LIM domains are boxed. Thebreaking point is indicated with an arrow.

[0239]FIG. 7

[0240] Nucleotide sequence if HMGI-C (U28749). The transcription startsite indicated as proposed by Manfioletti et al. [67] was arbitrarilychosen as a start site. The sequence contains the complete codingsequence.

[0241]FIG. 8

[0242] Gel of PCR products obtained as described in Example 5.

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[0324] ANNEX 1

Genes, Chromosome & Cancer 12:296-303 (1995)

[0325] Molecular Characterization of MAR, a Multiple Aberration Regionon Human Chromosome Segment 12q13-q15 Implicated in Various Solid Tumors

[0326] Wim J. M. Van de Ven, Eric F. P. M. Schoenmakers, SylkeWanschura, Bernd Kazmierczak, Patrick F. J. Kools, Jan M. W. Geurts,Sabine Bartnitzke, Herman Van den Berghe, and Jörn Bullerdiek

[0327] Center for Human Genetics, University of Leuven, Belgium(W.J.M.V.D.V., E.F.P.M.S., P.F.J.K., J.W.M.G., H.V.D.B.);

[0328] Center for Human Genetics, University of Bremen, Germany (S.W.,B.K., S.B., J.B.).

[0329] Chromosome arm 12q breakpoints in seven cell lines derived fromprimary pleomorphic salivary gland adenomas were mapped by FISH analysisrelative to nine DNA probes. These probes all reside in a 2.8 Mb genomicDNA region of chromosome segment 12q13-q15 and correspond to previouslypublished sequence-tagged sites (STS). Their relative positions wereestablished on the basis of YAC cloning and long range physical and STScontent mapping. The 12q breakpoints of five of the cell lines werefound to be mapping within three different subregions of the 445 kb DNAinterval that was recently defined as the uterine leiomyoma clusterregion of chromosome 12 breakpoints (ULCR12) between STS RM33 and RM98.All seven breakpoints appeared to map within the 1.7 Mb DNA regionbetween STS

[0330] RM36 and RM103. Furthermore, the chromosome 12 breakpoints ofthree primary pleomorphic salivary gland adenomas were also found to bemapping between RM36 and RM103. Finally, FISH analysis of two lipomacell lines with 12q13-q15 aberrations pinpointed the breakpoints ofthese to relatively small and adjacent DNA segments which, as well asthose of two primary lipomas, appeared to be located also between RM36and RM103. We conclude from the observed clustering of the 12qbreakpoints of the three distinct solid tumor types that the 1.7 Mb DNAregion of the long arm of chromosome 12 between RM36 and RM103 is amultiple aberration region which we designate MAR. Genes ChromosomCancer 12:296-303.(1995). @ 1995 Wiley-Liss, Inc.

Introduction

[0331] Chromosome translocations involving region q13-q15 of chromosome12 have been observed in a wide variety of solid tumors (Mitelman,1991). In subgroups of cytogenetically abnormal uterine leiomyomas(Nilbert and. Heim, 1990; Pandis et al., 1991), pleomorphic salivarygland adenomas (Sandros et al., 1990; Bullerdiek et al., 1993), andbenign adipose tissue tumors (Sreekantaiah et al., 1991), 12q13-q15aberrations are frequently observed. In a recent study (Schoenmakers etal., 1994b), we identified and molecularly characterized ULCR12, theuterine leiomyoma cluster region of chromosome 12 breakpoints. In thepresent study, we focus on the chromosome arm 12q breakpoints inpleomorphic adenoma of the salivary glands, a benign epithelial tumororiginating from the major or minor salivary glands. It is the mostcommon type of salivary gland tumor and accounts for almost 50% of allneoplasms in these organs. About 85% of the tumors are found in theparotid gland, 10% in the minor salivary glands, and 5% in thesubmandibular gland (Seifert et al., 1986). Although many of theseadenomas appear to have a normal karyotype, cytogenetic studies havealso revealed recurrent specific chromosome anomalies (Sandros et al.,1990; Bullerdiek et al., 1993). Besides chromosome 8 aberrations, oftentranslocations with a breakpoint in 8q12 with, as the most commonaberration, a t(3;8) (p21;q12), aberrations of chromosome 12, usuallytranslocations involving 12q13-q15, are also frequent. Non-recurrentclonal abnormalities have also been described. The frequent, involvementof region 12q13-q15 in distinct solid tumor types suggests that thischromosomal region harbors gene(s) that might be implicated in theevolution of these tumors. Molecular cloning of the chromosome 12breakpoints of these tumors and characterization of the junctionfragments may therefore lead to the identification of such gene(s).

[0332] On the basis of fluorescence in situ hybridization (FISH) data,we have previously reported that the chromosome 12 breakpoints in anumber of cell lines derived from primary pleomorphic salivary glandadenomas (Kazmierczak et al., 1990;.Schoenmakers et al., 1994a), arelocated on the long arm of chromosome 12 in the interval between lociD12S19 and D12S8 (Schoenmakers et al., 1994a). This DNA interval hasbeen estimated to be about 7 cM (Keats et al., 1989; Craig et al.,1993). The interval containing the chromosome 12 breakpoints of thesetumor cells was narrowed further by showing that all breakpoints mappeddistally to the CHOP gene, which is directly affected by thecharacteristic t(12;16) translocation in myxoid liposarcomas (Aman etal., 1992; Crozat et al., 1993; Rabbitts et al., 1993) and is locatedbetween D12S19 and D12S8. In more recent studies (Kools et al., 1995),the chromosome 12 breakpoint of pleomorphic salivary gland adenoma cellline Ad-312/SV40 was pinpointed to a DNA region between sequence-taggedsites (STSs) RM110 and RM111, which is less than 165 kb in size. FISHevaluation of the chromosome 12 breakpoints of the other pleomorphicsalivary gland adenoma cell lines indicated that they must be locatedproximally to the one in Ad-312/SV40, at a distance of more than 800 kb(Kools et al., 1995). These results pointed towards a possibledispersion of the chromosome 12 breakpoints over a relatively largegenomic region on the long arm of chromosome 12.

[0333] Here, we report physical mapping of the chromosome 12 breakpointsin pleomorphic salivary gland adenoma cells from primary tumors as wellas established tumor cell lines. The karyotypic anomalies observed inthe cells were all different but always involved region q13-q15 ofchromosome 12. Using DNA probes between D12S8 and CHOP, whichcorresponded to sequence-tagged sites (STSs) of a long-range physicalmap of a 6 Mb DNA region and were obtained during chromosome walkingexperiments, we performed FISH experiments and defined more precisely amajor chromosome 12 breakpoint cluster region of pleomorphic salivarygland adenoma. This breakpoint cluster region appeared to overlap withULCR12. Furthermore, we tested whether 12q13-q15 breakpoints of lipomasmight also map within the same region as those of pleomorphic salivarygland adenoma and uterine leiomyoma.

Materials and Methods

[0334] Primary Solid Tumors and Derivative Cell Lines.

[0335] Primary solid tumors including pleomorphic salivary glandadenomas, lipomas, and uterine leiomyomas were obtained from theUniversity Clinics in Leuven, Belgium (Dr. I. De Wever); in Bremen,Germany (Dr. R. Chille); in Krefeld, Germany (Dr. J. Haubrich); and fromthe Institute of Pathology in Göteborg, Sweden (Dr. G. Stenman). Forcell culturing and subsequent FISH analysis, tumor samples were finelyminced, treated for 4-6 hours with 0.8% collagenase (Boehringer,Mannheim, FRG), and processed further for FISH analysis according toroutine procedures.

[0336] Human tumor cell lines used in this study included the previouslydescribed pleomorphic salivary gland adenoma cell lines Ad-211/SV40,Ad-248/SV40, Ad-263/SV40, Ad-295/SV40, Ad-302/SV40, AD-366/SV40, andAd-386/SV40 (Kazmierczak et al., 1990; Schoenmakers et al., 1994a) andthe lipoma cell lines Li-14/SV40 (Schoenmakers et al., 1994a) andrecently developed Li-538/SV40. Chromosome 12 aberrations found in thesecell lines are listed in Table 1. Cells were propagated in TC199 culturemedium with Earle's salts supplemented with 20% fetal bovine serum.TABLE 1 Chromosome 12 Abberrations in Primary Human Solid Tumors andCell Lines* Aberration Cell lines Ad-211/SV40 t(8;12)(q21;q13-q15)Ad-248/SV40 ins(12;6)(q15;q16q21) Ad-263/SV40 inv(12)(q15q24.1)Ad-295/SV40 t(8:12;18)(p12;q14,p11.2) Ad-302/SV40 t(7;12)(q31;q14)Ad-366/SV40 inv(12)(p13q15) Ad-386/SV40 t(12;14)(q13-q15;q13-q15)Li-14/SV40 t(3;12)(q28;q13) Li-538/SV40 t(3;12)(q27;q14) LM-5.1/SV40t(12;15)(q15;q24) LM-30.1/SV40 t(12:14)(q15;q24) LM-65/SV40t(12;14)(q15;q24) LM-67/SV40 t(12;14)(q13-q15;q24) LM-100/SV40t(12:14)(q15;q24) LM-605/SV40 ins(12;11)(q14;q21qter) LM-608/SV40t(12;14)(q15;q24) LM-609/SV40 t(12;14)(q15;q24) Primary rumors Ad-386t(12;14)(q15;q11.2) Ad-396 t(3;12) Ad-400 t(12;16) Li-166 t(12;12)Li-167 t(3;12)(q28;q14-q15) LM-163.1 t(12;14)(q14;q24) LM-163.2t(12;14)(q14-q24) LM-168.3 t(X;12)(q22:q15) LM-192t(2;3;12)(q35;p21;q14) LM-196.4 t(12;14)(q14;q24)

DNA Probes

[0337] In the context of a human genome project focusing on the long armof chromosome 12, we isolated cosmid clones cRM33, cRM36, cRM51, cRM69,cRM72, cRM76, cRM98, cRM103, and cRM133, from chromosome 12-specificarrayed cosmid library LLNL12NC01 (Montgomery et al., 1993). Furtherdetails of these cosmid clones have been reported at the SecondInternational Chromosome 12 Workshop (1994) and will be describedelsewhere (Kucherlapati et al., 1994). Briefly, initial screenings wereperformed using a PCR-based screening strategy (Green and Olson, 1990),followed by filter hybridization analysis as the final screening step,as previously described (Schoenmakers et al., 1994b). The cosmid cloneswere isolated using STSs derived from YAC clones. STSs were obtainedupon rescue of YAC insert-ends using a methodology involvingvectorette-PCR followed by direct solid phase fluorescent sequencing ofthe PCR products (Geurts et al., 1994) or from inter-Alu PCR (Nelson etal., 1989). Cosmid clones were grown and handled according to standardprocedures (Sambrook et al., 1989).

[0338] Cosmid clone cPK12qter, which maps to the telomeric region of thelong arm of chromosome 12 (Kools et al., 1995) was used as a referencemarker.

[0339] Chromosome Preparations and Fluorescence In Situ Hybridization.

[0340] Metaphase spreads of the pleomorphic salivary gland adenoma celllines or normal human lymphocytes were prepared as described before(Schoenmakers et al., 1993). To unambiguously establish the identity ofchromosomes in the FISH experiments, FISH analysis was performed afterGTG-banding of the same metaphase spreads. GTG-banding was performedessentially as described by Smit et al. (1990). In situ hybridizationswere carried out according to a protocol described by Kievits et al.(1990) with some minor modifications (Kools et al., 1994; Schoenmakerset al., 1994b). Cosmid and YAC DNA was labelled with biotin-11-dUTP(Boehringer Mannheim) or biotin-14-dATP (BRL, Gaithersburg) as describedbefore (Schoenmakers et al., 1994b). Specimens were analyzed on a ZeissAxiophot fluorescence microscope using a FITC filter (Zeiss). Resultswere recorded on Scotch (3M) 640 asa film.

Results

[0341] FISH Mapping of 12q Breakpoints in Cell Lines of PleomorphicSalivary Gland Adenoma.

[0342] In previous studies (Schoenmakers et al., 1994a), we mapped thechromosome 12 breakpoints in a number of pleomorphic adenomas of thesalivary glands relative to various DNA markers and established thatthese were all located proximally to locus D12S8 and distal to the CHOPgene. This region is somewhat smaller than the 7 cM region encompassedby linkage loci D12S8 and D12S19 (Keats et al., 1989). Using YACcloning, a long range physical/STS map has been constructed coveringmost of that 7 cM region, as recently reported (Kucherlapati et al.,1994). Furthermore, numerous genomic clones (cosmid clones) have beenisolated and their relative positions within this map established(Kucherlapati et al., 1994). Nine of these cosmids, including cRM33,cRM36, cRM5l, cRM69, cRM72, cRM76, cRM98, cRM103, and cRM133, were usedin FISH studies to establish the positions of the chromosome 12breakpoints of the seven cell lines derived from pleomorphic adenomas ofthe salivary glands (Table 1). The relative mapping order of these ninecosmid clones, which cover a genomic region on the long arm ofchromosome 12 of about 2.8 Mb, is indicated in FIG. 1 and the results ofFISH studies with the various cosmid probes are schematically summarizedin the same figure. As an illustration, FISH results obtained withmetaphase cells of cell line Ad-295/SV40 using cRM76 and cRM103 asprobes are shown in FIG. 2. It should be noted that for theidentification of chromosomes, pre-FISH GTG-banding was used routinely.On the basis of such banding, hybridization signals could be assignedconclusively to chromosomes of known identity; this was of majorimportance for cases with cross- or background hybridization signals, asthese were occasionally observed. When GTG-banding in combination withFISH analysis provided inconclusive results, either because of weakhybridization signals or rather vague banding, FISH experiments wereperformed with cosmid clone cPK12ater (Kools et al., 1995) as areference probe.

[0343] FISH analysis of metaphase chromosomes of each of the sevenpleomorphic salivary gland adenoma cell lines with cosmid cRM103revealed that this cosmid mapped distal to the chromosome 12 breakpointsof all seven cell lines studied here. Metaphase chromosomes of six ofthe seven cell lines were also tested with probe cRM69 and, in twocases, with cRM51. The results of the latter experiments were alwaysconsistent with those obtained with cRM103. Similar FISH analysis withcRM36 as probe indicated that this probe mapped proximal to all thebreakpoints. These results were always consistent with those obtainedfor five of the seven cell lines in experiments using cRM72. Altogether,the results of our FISH studies indicated that the chromosome 12breakpoints of all seven cell lines map between cRM36 and cRM103, whichspans a genomic region of about 1.7 Mb.

[0344] Fine Mapping of 12q Breakpoints in Cell Lines Derived FromPleomorphic Adenomas of the Salivary Glands.

[0345] For subsequent fine mapping of the chromosome 12 breakpoints ofthe seven pleomorphic salivary gland adenoma cell lines, additional FISHstudies were performed, as schematically summarized in FIG. 1. Thebreakpoints of cell lines Ad-211/SV40, Ad-295/SV40, and Ad-366/SV40appeared to be located in the DNA region between cRM76 and cRM133, whichwas estimated to be about 75 kb. The breakpoints of the four other celllines were found in different areas of the 1.7 Mb region between cRM36and cRM103. That of cell line Ad-248/SV40 in a DNA segment of about 270kb between cRM33 and cRM76, that of Ad-263/SV40 in a DNA segment ofabout 1 Mb between cRM98 and cRM103, that of Ad-302/SV40 in a DNAsegment of about 240 kb between cRM33 and cRM36, and that of Ad-386/SV40in a DNA segement of about 100 kb between cRM98 and cRM133. Inconclusion, these results indicated that the chromosome 12 breakpointsof most (5 out of 7) of the cell lines are dispersed over the 445 kbgenomic region on the long arm of chromosome 12 between cRM33 and cRM98.It is important to note already here that precisely this region wasrecently shown to contain the chromosome 12q breakpoints in cell linesderived from primary uterine leiomyomas (see FIG. 3) and was thereforedesignated ULCR12 (Schoenmakers et al., 1994b). As this segment of thelong arm of chromosome 12 is involved in at least two types of solidtumors (Schoenmakers et al., 1994b; this study) and, as we will showbelow, also in a third solid tumor type, we will from now on refer tothe DNA interval between cRM36 and cRM103 as MAR (multiple aberrationregion).

[0346] FISH Mapping of 12q Breakpoints in Primary Pleomorphic SalivaryGland Adenomas.

[0347] Our FISH studies on metaphase chromosomes of pleomorphic adenomasof the salivary glands presented so far were restricted to cell linesderived from primary tumors. Although it is reasonable to assume thatthe chromosome 12 breakpoints in cell lines are similar if not identicalto the ones in the corresponding primary tumors, differences as a resultof the establishment of cell lines or subsequent cell culturing cannotfully be excluded. Therefore, we have investigated whether thechromosome 12 breakpoints in three primary salivary gland adenomas weremapping to MAR as well. To test this possibility, a combination ofcosmid clones cRM33 and cRM103 were used as molecular probe. In allthree cases, this cosmid pool clearly spanned the chromosome 12breakpoints (data not shown), indicating that these breakpoints wereindeed localized within MAR. In a recent study (Wanschura et al.,submitted for publication), it was reported that the chromosome-12breakpoints of five primary uterine leiomyomas with 12q14-15 aberrationswere all found to cluster within the 1.5 Mb DNA fragment (between cRM33and cRM103), which is known to harbor the breakpoints of various celllines derived from primary uterine leiomyomas (schematically summarizedin FIG. 3). Consistent with the results of the breakpoint mappingstudies using cell lines, the results with the two primary solid tumortypes establish that the breakpoints of the primary tumor cells arelocated in MAR.

[0348] Chromosome Segment 12q13-q15 Breakpoints of Lipomas Mappingwithin MAR.

[0349] To test the possibility that the chromosome 12 breakpoints ofother solid tumors with 12q13-q15 aberrations also mapped within MAR, westudied two lipomas cell lines by FISH analysis—Li-14/SV40 andLi-538/SV40. The chromosome 12 aberrations of these two lipoma celllines are given in Table 1. As molecular probes, cosmid clones cRM33,cRM53, cRM72, cRM76, cRM99, cRM103, and cRM133 were used. The breakpointof Li-14/SV40 was mapped to the 75 kb DNA interval between RM76 andRM133, and that of Li-538/SV40 to the 90 kbp interval between RM76 andRM99 (data not shown), as schematically illustrated in FIG. 3. SimilarFISH analysis of two primary lipomas using a mixture of cRM36 and cRM103as molecular probe resulted in a hybridization pattern indicating thatthe mixture of probes detected sequences on either side of thebreakpoints. These results are the first indications that also inlipoma, chromosome 12q13-q15 breakpoints occur that map within MAR. Morelipoma cases should be tested to allow proper interpretation of thisobservation.

Discussion

[0350] In this study, we have mapped the chromosome 12 breakpoints ofthree primary pleomorphic salivary gland adenomas as well as sevenestablished cell lines derived from such tumors. All breakpointsappeared to be located in a previously molecularly cloned andcharacterized chromosome DNA segment on the long arm of chromosome 12,of about 1.7 Mb in size, with five of them clustering in a DNA intervalof less than 500 kb. The 1.7 Mb DNA region apparently contains a majorbreakpoint cluster region for this type of tumor. In a previous study,we have described the characterization of the chromosome 12 breakpointof pleomorphic salivary gland adenoma cell line Ad-312/SV40 (Kools etal., 1995). The breakpoint of this cell line is now known to map at adistance of more than 2 Mb distally to this major breakpoint clusterregion reported here. It is possible that the Ad-312/SV40 breakpointinvolves other pathogenetically relevant genetic sequences than thoseaffected by the clustered breakpoints. However, the possibility shouldnot yet be excluded that all the 12q13-q15 breakpoints in pleomorphicsalivary gland adenomas mapped so far belong to the same category andare dispersed over a relatively large DNA region of this chromosome,reminiscent of the 11q13 breakpoints in B-cell malignancies (Raynaud etal., 1993). More precise pinpointing of the various breakpoints couldshed more light on this matter.

[0351] Of importance is the observation that the DNA segment thatharbors the clustered 12q breakpoints of pleomorphic salivary glandadenomas appears to coincide with the DNA region that was recentlydefined as the uterine leiomyoma cluster region of chromosome 12breakpoints, known as ULCR12 (Schoenmakers et al., 1994b). Of furtherinterest is the fact that this region of chromosome 12 also harborsbreakpoints of primary lipomas and lipoma cell lines derived fromprimary tumors with 12q13-q15 aberrations. Altogether, the results ofall these studies now clearly demonstrate that chromosome 12 breakpointsof three distinct solid tumor types map to the same 1.7 Mb genomicregion on the long arm of chromosome 12, establishing this region to bea multiple aberration region. To reflect this characteristic, we havedesignated this DNA segment MAR.

[0352] Genetic aberrations involving chromosomal region 12q13-q15 havebeen implicated by many cytogenetic studies in a variety of solid tumorsother than the three already mentioned. Involvement of 12q13-q15 hasalso been reported for endometrial polyps (Walter et al., 1989; Vanni etal., 1993), clear cell sarcomas characterized by recurrent t(12;22)(q13;q13) (Fletcher, 1992; Reeves et al., 1992; Rodriguez et al., 1992),a subgroup of rhabdomyosarcoma (Roberts et al., 1992) andhemangiopericytoma (Mandahl et al., 1993a), chondromatous tumors(Mandahl et al., 1989; Bridge et al., 1992; Hirabayashi et al., 1992;Mandahl et al., 1993b), and hamartoma of the lung (Dal Cin et al.,1993). Finally, several case reports of solid tumors with involvement ofchromosome region 12q13-q15 have been published—e.g., tumors of thebreast (Birdsal et al., 1992; Rohen et al., 1993), diffuse astrocytomas(Jenkins et al., 1989), and a giant-cell tumor of the bone (Noguera etal., 1989). On the basis of results of cytogenetic studies, nopredictions could be made about the relative distribution of thebreakpoints of these tumor types. In light of the results of the presentstudy, it would be of interest to see whether the breakpoints of any ofthese solid tumors also map within or close to MAR. The various cosmidclones available now provide the means to test this readily.

[0353] The observation that 12q breakpoints of at least three differenttypes of solid tumors map to the same DNA region is intriguing as itcould be pointing towards the possibility that the same geneticsequences in MAR are pathogenetically relevant for tumor development indifferent tissues. If so, it is tempting to speculate that the gene(s)affected by the genetic aberrations might be involved in growthregulation. On the other hand, one cannot yet exclude the possibilitythat genetic sequences in MAR are not pathogenetically relevant, as theobserved clustering of genetic aberrations in MAR could simply reflectgenetic instability of this region, which becomes apparent in varioussolid tumors. To obtain more insight in this matter, the genes residingin MAR should be identified and characterized, and this can be achievedby various approaches using several techniques (Parrish and Nelson,1993).

Acknowledgments

[0354] The constructive support of managing director G. Everaerts isgreatly acknowledged. The authors would like to thank P. Dal Cin, J.Haubrich, R. Hille, G. Stenman, and I. De Wever for providing the solidtumor specimens studied in the present report; C. Huysmans, E. Meyen, K.Meyer-Bolte, R. Mols, and M. Willems for excellent technical assistance;and M. Leys for artwork. This work was supported in part by the ECthrough Biomed 1 program “Molecular Cytogenetics of Solid Tumours”, the“Geconcerteerde Onderzoekacties 1992-1996”, the National Fund forScientific Research (NFWO; Kom op tegen Kanker), the “ASLK-programmavoor Kankeronderzoek”, the “Schwerpunktprogramm: Molekulare undKlassische Tumorcytogenetik” of the Deutsche Forschungsgemeinschaft, andthe Tönjes-Vagt Stiftung. This text presents results of the Belgianprogramme on Interuniversity Poles of attraction initiated by theBelgian State, Prime Minister's Office, Science Policy Programming. Thescientific responsibility is assumed by its authors. J. W. M. Geurts isan “Aspirant” of the National Fund for Scientific Research (NFWO; Kom optegen Kanker).

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[0400] Wanschura S, Belge G, Stenman G, Kools P, Dal Chin P.Schoenmakers E, Huysmans C, Bartnizke S, Van de Ven W, and Bullerdiek J(submitted for publication). Mapping of the translocation breakpoints ofprimary pleomorphic adenomas and lipomas within a common region ofchromosome 12.

[0401] Legends of Figures of ANNEX 1

[0402]FIG. 1. Schematic representation of FISH mapping data obtained forthe seven pleomorphic salivary gland adenoma cell lines tested in thisstudy. Cosmid clones which were used as probes in the FISH mappingstudies map at sequence-tagged sites obtained from overlapping YACclones. They are named after the acronyms of the STSs, as shown in theboxes, and the relative order of these is as presented. The DNA intervalbetween RM69 and RM72 is estimated to be about 2.8 Mb. The solid linesindicate DNA intervals in which the breakpoints of the various celllines are located. The dots indicate FISH experiments that wereperformed on metaphase chromosomes of the various cell lines using acosmid clone corresponding to the STS indicated above these as molecularprobe. The relative positions of MAR and ULCR12 are indicated in thelower part of the figure. Ad, pleomorphic salivary gland adenoma; MAR,multiple aberration region; ULCR12, uterine leiomyoma cluster region ofchromosome 12 breakpoints.

[0403]FIG. 2. a: Partial karyotype of Ad-295/SV40 showing der(8),der(12), der(18) and the corresponding normal chromosomes. b: FISHanalysis of metaphase chromosomes of Ad-295/SV40 cells using DNA ofcosmid clone cRM76 as molecular probe. Hybridization signals on normalchromosome 12 (arrow) and der(12) (arrowhead). c: GTG-banding pattern ofmetaphase chromosomes of Ad-295/SV40 shown in b. d: FISH analysis ofmetaphase chromosomes of Ad-295/SV40 cells using DNA of cosmid clonecRM103 as molecular probe. Hybridization signals on normal chromosome 12(arrow) and der(18) (arrowhead).

[0404]FIG. 3. Schematic representation of chromosome 12 breakpointmapping data obtained for primary pleomorphic salivary gland adenomas,uterine leiomyomas, and lipomas as well as cell lines derived from suchsolid tumors. Results are compared to data for primary uterineleiomyomas (Wanschura et al., submitted for publication) and cell linesderived from such tumors (Schoenmakers et al., 1994b). Cosmid cloneswhich were used as probes in the FISH mapping studies correspond tosequence-tagged sites obtained from overlapping YAC clones. Cosmidclones were named after the acronyms of the STSs, as shown in the boxes,and the relative order of these is as presented. The estimated sizes ofDNA intervals between STSs are indicated. Ad, pleomorphic salivary glandadenoma; Li, lipoma; LM, uterine leiomyoma.

[0405] ANNEX 2

[0406] Lead Article

[0407] Identification of the Chromosome 12 Translocation BreakpointRegion of a Pleomorphic Salivary Gland Adenoma with t(1;12) (p22;q15) asthe Sole Cytogenetic Abnormality

[0408] Patrick F. J. Kools, Sylke Wanschura, Eric F. P. M. Schoenmakers,Jan W. M. Geurts, Raf Mols, Bernd Kazmierczak, Jörn Bullerdiek, HermanVan den Berghe and Wim J. M. Van de Ven

[0409] ABSTRACT: Cell line Ad-312/SV40, which was derived from a primarypleomorphic salivary gland adenoma with t(1;12)(p22;q15), was used influorescence in situ hybridization (FISH) analysis to characterize itstranslocation breakpoint region on chromosome 12. Results of previousstudies have indicated that the chromosome 12 breakpoint in Ad-312/SV40is located proximally to locus D12S8 and distally to the CHOP gene. Wehere describe two partially overlapping yeast artificial chromosome(YAC) clones, Y4854 (500 kbp) and Y9091 (460 kbp), which we isolated inthe context of a chromosome walking project with D12S8 and CHOP asstarting points. Subsequently, we have isolated cosmid clonescorresponding to various sequence-tagged sites (STSs) mapping within theinserts of these YAC clones. These included cRM51, cRM69, cRM85, cRM90,cRM91, cRM110, and cRM111.

[0410] We present a composite long-range restriction map encompassingthe inserts of these two YAC clones and show by FISH analysis, that bothYACs span the chromosome 12 breakpoint as present in Ad-312/SV40 cells.In FISH studies, cosmid clones cRM85, cRM90 and cRM111 appeared to mapdistally to the chromosome 12 breakpoint whereas cosmid clones cRM51,cRM69, cRM91, and cRM110 were found to map proximally to it. Theseresults assign the chromosome 12 breakpoint in Ad-312/SV40 to a DNAregion of less than 165 kbp. FISH evaluation of the chromosome 12breakpoints in five other pleomorphic salivary gland adenoma cell linesindicated that these are located proximally to the one in Ad-312 SV40,at a distance of more than 0.9 Mb from STS RM91. These results, whilepinpointing a potentially critical region on chromosome 12, also provideevidence for the possible involvement of chromosome 12q13-q15 sequenceslocated elsewhere.

[0411] Introduction

[0412] Pleomorphic salivary gland adenoma constitutes a benignepithelial tumor that originates from the major and minor salivaryglands. It is the most common type of salivary gland tumor and accountsfor almost 50% of all neoplasms in these organs; 85% of the tumors arefound in the parotid gland, 10% in the minor salivary glands, and 5% inthe submandibular gland [1]. About 50% of these adenomas appear to havea normal karyotype but cytogenetic studies have also revealed recurrentspecific chromosome anomalies [2, 3]. Frequently observed anomaliesinclude aberrations of chromosome 8, usually involving the 8q12-q13region, with the most common aberration a t(3;8) (p21;q12), andaberrations of chromosome 12, usually translocations involving region12q13-q15. Non-recurrent clonal chromosome abnormalities have also beenreported. The highly specific pattern of chromosome rearrangements withconsistent breakpoints at 8q12-q13 and 12q13-q15 suggests that thesechromosomal regions harbour genes that might be implicated in thedevelopment of these tumors. Molecular cloning of the chromosomebreakpoints and characterization of their junction fragments may lead tothe identification of pathogenetically relevant genes. At present, nosuch molecular data have yet been reported for these tumors.

[0413] On the basis of fluorescence in situ hybridization (FISH) data,the chromosome 12 breakpoints in six pleomorphic salivary gland adenomacell lines were recently shown to be mapping to region 12q13-q15, moreprecisely, to the genomic interval between loci D12S19 and D12S8 [4, 5].The sex-averaged genetic size of this genomic DNA interval was reportedat HGM10 to be 7 cM [6]. We also reported that the chromosome 12breakpoints in salivary gland adenomas map distally to the CHOP gene[5], which supports an earlier study indicating that the 12q13-q15translocation breakpoints in pleomorphic salivary gland adenomas aredifferent from that in myxoid liposarcoma [7]. Here, we report about thephysical mapping of the chromosome 12 breakpoint in pleomorphic salivarygland adenoma cell line Ad-312/SV40, which carries a t(1;12)(p22;q15) asthe only cytogenetic abnormality.

[0414] Materials and Methods

[0415] Tumor Cell Lines.

[0416] Human tumor cell lines used in this study included the previouslydescribed pleomorphic salivary gland adenoma cell lines Ad-248/SV40,Ad-263/SV40, Ad-295/SV40, Ad-302/SV40, Ad-312/SV40, and Ad-366/SV40 [5,8]. Cells were cultivated in TC199 culture medium with Earle's saltssupplemented with 20% fetal bovine serum. Other cell lines used in thisstudy included somatic cell hybrid PK89-12, which contains chromosome 12as the sole human chromosome in a hamster genetic background [9], andsomatic cell hybrid LIS-3/SV40/A9-B4 [4]. The latter cell line wasobtained upon fusion of myxoid liposarcoma cell line LIS-3/SV40, whichcarries the specific t(12;16)(q13;p11.2), with mouse A9 cells. Thissomatic cell hybrid was previously shown to contain der(16) but neitherder(12) nor the normal chromosome 12 [4]. PK89-12 and LIS-3/SV40/A9-B4cells were grown in DME-F12 medium supplemented with 10% fetal bovineserum. Cell lines were analyzed by standard cytogenetic techniques atregular intervals.

[0417] Isolation of YAC and Cosmid Clones.

[0418] In the context of human genome mapping studies, which will bedescribed in detail elsewhere (Schoenmakers et al., in preparation), weisolated YAC clones Y4854 and Y9091 from the first-generation CEPH YAClibrary [10], and cosmid clones cRM51, cRM69, cRM85, cRM90, cRM91,cRM103, cRM110, and cRM111 from the chromosome-12-specific, arrayedcosmid library LLNLNC01 [11]. YAC and cosmid clones were isolated asdescribed before [5]. Initial screenings of the YAC, as well as thecosmid library, were performed using a screening strategy, involving thepolymerase chain reaction (PCR) [12]. Filter hybridization analysis wasused as the final screening step, as previously described [5]. Cosmidclones were isolated using STSs and those corresponding to STSs withinthe inserts of YAC clones Y4854 and Y9091 are indicated in FIG. 1. STSswere obtained via rescue of YAC insert end-sequences using avectorette-PCR procedure [13] or Alu-PCR [14, 15]. PCR products weresequenced directly via solid-phase fluorescent sequencing. Cosmid cloneswere grown and handled according to standard procedures [16]. YAC cloneswere characterized by pulsed-field gel electrophoresis [17], restrictionmapping, and hybridization, as previously described [5].

[0419] Chromosome Preparations and Fluorescence In Situ Hybridization.

[0420] Cells from the pleomorphic salivary gland adenoma tumor celllines were treated with Colcemid (0.04 μg/ml) for 30 min and thenharvested according to routine methods. Metaphase spreads of the tumorcells were prepared as described before [4]. To establish the identityof chromosomes in the FISH experiments, FISH analysis was performedafter G-banding of the same metaphase spreads. G-banding was performedessentially as described by Smit et al. [18]. In situ hybridizationswere carried out according to a protocol described by Kievits et al.[19] with some minor modifications [5, 20]. Cosmid and YAC DNA waslabelled with biotin-11-dUTP (Boehringer Mannheim) or biotin-14-dATP(BRL, Gaithersburg), as described earlier [5]. Chromosomes werecounterstained with propidium iodide and analyzed on a Zeiss Axiophotfluorescence microscope using a FITC filter (Zeiss). Results wererecorded on Scotch (3M) 640 asa film.

[0421] Results

[0422] Isolation and Characterization of YAC Clones Spanning theChromosome 12 Breakpoint of Pleomorphic Salivary Gland Adenoma Cell LineAd-312/SV40.

[0423] In previous studies [5], we mapped the chromosome 12 breakpointsof six pleomorphic salivary gland adenoma cell lines proximally to locusD12S8 and distally to CHOP. The DNA interval between these loci issomewhat smaller than 7 cM (estimated distance between the loci D12S8and D12S19 [6]) but still substantially large. To molecularly define thetranslocation breakpoint of Ad-312/SV40, we have performed human genomemapping studies on the DNA interval between locus D12S8 and the CHOPgene. In the process of directional chromosome walking starting fromD12S8 and the CHOP gene, we obtained overlapping YAC clones Y9091 andY4854. The DNA insert of Y9091 appeared to be 460 kbp and that of Y4854,500 kbp. Moreover, as we will demonstrate below, the DNA insert of eachYAC clone appeared to span the chromosome 12 breakpoint of Ad-312/SV40.A long-range restriction map of the inserts of these YAC clones was madeusing pulsed-field gel electrophoresis and hybridization analysis (FIG.1). On the basis of STS content mapping and Southern blot analysis, theinserts of YAC clones Y9091 and Y4854 appeared to overlap as indicatedin FIG. 1. The tested STSs correspond to end-sequences of otheroverlapping YAC clones not shown here or to sequences obtained viainter-Alu-PCR. Of these, RM90 and RM91 represent such end-clone STSs ofYAC Y9091, and RM48 and RM54 of Y4854, whereas RM110 and RM111 representSTSs derived from inter-Alu-PCR. For a number of STSs mapping within theinserts of YAC clones Y4854 and Y9091, corresponding cosmid clones wereisolated for use in FISH analysis, e.g., cRM51, cRM69, cRM85, cRM90,cRM91, cRM110, and cRM111.

[0424] The inserts of the two overlapping YAC clones are most likely notchimeric, as was deduced from the following observations. FISH analysisof metaphase chromosomes of normal human lymphocytes with Y4854 or Y9091DNA as molecular probe revealed hybridization signals only in chromosomeregion 12q13-q15. For Y9091, this was confirmed further by observationsmade in FISH studies in which cosmid clone cRM90 or cRM91 was used asprobe; the DNA insert of each of these two cosmids corresponds to thealternative end-sequences of YAC clone Y9091. Finally, the end-sequenceSTSs of Y9091 appeared to map to chromosome 12 and distally to the CHOPgene, as was established by PCR analysis on PK89-12 DNA,; which containshuman chromosome 12 as the sole human chromosome in a hamster geneticbackground, and LIS-3/SV40/A9-B4 DNA, which was previously shown tocontain der(16), from the specific t(12;16) of myxoid liposarcoma, butneither der(12) nor the normal chromosome 12 [4]. From the chromosomewalking studies, we concluded that the overlapping inserts of the twoYAC clones represent a DNA region of about 640-kbp, which is located onchromosome 12q between D12S8 and CHOP. As the 640-kbp compositelong-range restriction map of the YAC contig was constructed with atleast double coverage of the entire region, it is not unreasonable toassume that the 640-kbp region is contiguous with the chromosomal DNA,although microdeletions can not be excluded at this point.

[0425] Chromosome walking was routinely evaluated by FISH mapping of YACclones and/or cosmid clones corresponding to YAC insert sequences. Itshould be noted that for the identification of chromosomes, G-bandingwas used in most cases. On the basis of such G-banding, hybridizationsignals could be assigned conclusively to chromosomes of known identity;this was also of importance for the cases with cross- or backgroundhybridization signals that were occasionally observed. G-banding priorto FISH analysis resulted sometimes in rather weak hybridization signalsor rather vague banding patterns. Therefore, we performed FISHexperiments in which the YAC and cosmid clones to be evaluated were usedin combination with a reference probe. Cosmid clone cPK12qter, which wasserendipitously obtained during screening of a cosmid library, wasselected as reference marker. FISH analysis of metaphase chromosomes ofnormal lymphocytes (FIG. 2A) revealed that cPK12gter maps to thetelomeric region of the long arm of chromosome 12. To identifychromosome 12 in this experiment, centromere 12-specific probe pα12H8[21] was used. FISH analysis of metaphase chromosomes of Ad-312/SV40cells using YAC clone Y4854 (FIG. 2B) or Y9091 (FIG. 2C) in combinationwith reference probe cPK12qter revealed, in both cases, hybridizationsignals of the YAC insert on der(1) as well as der(12). We concludedfrom these results that the insert DNA of each YAC clone might span thechromosome 12 breakpoint in this cell line. It should be noted thatG-banding revealed a telomeric association involving the short arm ofchromosome 12 in FIG. 2C. The observation that YAC clone Y9091 spannedthe chromosome 12 breakpoint in Ad-312/SV40 was confirmed independentlyin FISH studies in which cosmid clone cRM90 or cRM91 was used asmolecular probe; they were shown to contain the alternativeend-sequences of the Y9091 insert. cRM90 appeared to map distally to thechromosome 12 breakpoint, whereas cRM91 was found to map proximally(data not shown). These results also established the chromosomalorientation of the YAC contig shown in FIG. 1. In summary, we concludedfrom these FISH studies that the chromosome 12 translocation breakpointin Ad-312/SV40 must be located in the DNA interval corresponding to theoverlapping sequences (about 300 kbp) of the two YAC clones.

[0426] Fine Mapping of the Chromosome 12 Translocation Breakpoint ofAd-312/SV40.

[0427] In an approach to further narrow the chromosome 12 translocationbreakpoint region of Ad-312/SV40, cosmid clones with different mappingpositions within YAC clone Y9091 were isolated. These included cRM69,cRM85, cRM110, and cRM111. cRM69 and cRM85 were isolated on the basis ofSTS sequences of YAC clones not shown here. cRM110 and cRM111 wereobtained via inter-Alu-PCR. RM110 was shown by Southern blot analysis tohybridize to a terminal MluI fragment of Y9091 and not to the DNA insertof the overlapping YAC clone with RM69 as telomeric end-sequences. Thelocation of RM110 is as indicated in FIG. 1. RM111 was shown tohybridize to a BssHII, MluI, PvuI, and SfiI fragment of Y9091 and istherefore located in the PvuI-SfiI fragment of Y9091, to which STS RM48was also mapped (FIG. 1). FISH analysis of metaphase chromosomes ofAd-312/SV40 with cRM69 or cRM110 as probe indicated that the DNA insertof these cosmids mapped proximally to the chromosome 12 translocationbreakpoint in this cell line, as illustrated for cRM69 in FIG. 3A.Subsequent FISH analysis of Ad-312/SV40 with cRM85 or cRM111 as proberevealed hybridization signals distally to the translocation breakpoint,as illustrated for cRM111 in FIG. 3B. The results with cRM85 and cRM111are in agreement with the observed breakpoint spanning by YAC cloneY4854 as cRM85 maps distally and cRM111 closely to STS RM48, which marksthe telomeric end of YAC clone Y4854. In conclusion, the chromosome 12translocation breakpoint in Ad-312/SV40 must be located in the DNAinterval between cRM110 and cRM111, as schematically summarized in FIG.4.

[0428] FISH Evaluation of Chromosome 12 Breakpoints in Other PleomorphicSalivary Gland Adenoma Cell Lines.

[0429] To determine the position of their chromosome 12 breakpointsrelative to that of Ad-312/SV40, five other pleomorphic salivary glandadenoma cell lines were evaluated by FISH analysis, as summarizedschematically in FIG. 4. These cell lines, which were developed fromprimary tumors [5, 8], included Ad-248/SV40, Ad-263/SV40, Ad-295/SV40,Ad-302/SV40, and Ad-366/SV40. The chromosome 12 aberrations of thesecell lines are listed in FIG. 4. FISH analysis of metaphase chromosomesof these cell lines using cRM91 revealed that the chromosome 12breakpoints of all these cell lines mapped proximally to this cosmidclone (data not shown). Similar FISH analysis was also performed using acosmid clone corresponding to sequence-tagged site RM103 as a probe.RM103 was found to map proximally to RM91 at a distance of about 0.9Mbp. In all cases, cRM103 appeared to map distally to the chromosome 12translocation breakpoints, indicating that the chromosome 12 breakpointsin these five pleomorphic salivary gland adenoma cell lines are locatedat a relatively large distance from that of Ad-312/SV40 cells.

[0430] Discussion

[0431] In the studies presented here, we have identified, molecularlycloned, and characterized a chromosome region on the long arm ofchromosome 12 in which the translocation breakpoint of pleomorphicsalivary gland adenoma cell line Ad-312/SV40 appears to map. In previousstudies [5], we already provided evidence that the chromosome 12breakpoint of this cell line was located between D12S8 and CHOP. Becausethe two breakpoints spanning YAC clones described here were obtained indirectional chromosome walking experiments using D12S8 and the CHOP geneas initial starting points, the chromosome 12 breakpoint mapping resultspresented here confirm our previous claim. The FISH results obtainedwith the complete YAC insert of Y9091 as molecular probe were confirmedindependently in FISH studies using cosmid clones containing sequencescorresponding to various regions of the insert of this YAC clone. Thisis of importance, as the independent confirmatory results make it ratherunlikely that the split signals observed with the complete insert ofY9091 can be explained otherwise than by a factual splitting ofsequences represented in the YAC. The presence, for instance, of highlyrelated genetic sequences on both sides of a chromosome breakpoint couldeasily lead to erroneous conclusions if they were based solely on FISHresults of a YAC insert. Finally, our mapping studies have alsoestablished conclusively the chromosomal orientation of the long-rangerestriction map we have generated in these studies. This orientation wasalready predicted on the basis of two-color FISH studies (unpublishedobservations).

[0432] The FISH studies, described here, enabled us to map thechromosome 12 breakpoint in Ad-312/SV40 cells to the 190-kbp DNAinterval between the established STSs RM48 and RM69. However, thebreakpoint region can be narrowed somewhat further on the basis of thefollowing. The fact that Y4854 was shown to span the breakpointindicates that at least a considerable part of the telomeric half ofthis YAC clone must map distally to the breakpoint. Precisely how muchremains to be established. On the other side, STS RM69 appeared to belocated in about the middle of the DNA insert of cosmid clone cRM69,suggesting that the breakpoint is close to 25 kbp distally to RM69.Moreover, cRM69 appeared to lack RM110 (data not shown) and, as cRM110was found proximally to the chromosome 12 breakpoint in Ad-312/SV40cells, the breakpoint should be even further distal to RM69 than theearlier-mentioned 25 kbp. Altogether, this narrows the chromosome 12breakpoint region to a DNA interval, which must be considerably smallerthan 165 kbp. Further pinpointing of the breakpoint will allow us tomolecularly clone the chromosome 12 breakpoint and to characterize thegenetic sequences in the breakpoint junction region, which might lead tothe identification of pathogenetically relevant sequences.Identification of the genes present in the DNA inserts of YAC clonesY4854 and Y9091, via sequencing, direct hybridization, direct selectionor exon-trapping, might constitute a useful alternative approach foridentifying the gene in this region of the long arm of chromosome 12that might be pathogenetically critical for pleomorphic salivary glandadenoma tumorigenesis.

[0433] The observation that the chromosome 12 breakpoints in otherpleomorphic salivary gland adenomas are located in a remote and moreproximal region on the long arm of chromosome 12 is of interest. Itcould imply that the chromosome 12 breakpoints in pleomorphic salivarygland adenomas are dispersed over a relatively large DNA region of thelong arm of chromosome 12, reminiscent to the 11q13 breakpoints inB-cell malignancies [22]. Elucidation of the precise location of thechromosome 12 breakpoints in the other pleomorphic salivary glandadenoma cell lines could shed more light on this matter on the otherhand, it could point towards alternative sequences on the long arm ofchromosome 12 between D12S8 and the CHOP gene that might be ofimportance, presumably for growth regulation in pleomorphic salivarygland adenoma. The fact that the chromosome 12 breakpoint regiondescribed here has sofar been found only in the Ad-312/SV40 cell linemakes it necessary to analyze a larger number of salivary gland adenomaswith chromosome 12q13-q15 aberrations to assess the potential relevancefor tumorigenesis of the chromosome 12 sequences affected in the studiedcell line. If more cases with aberrations in this particular region ofchromosome 12 can be found, it would be of interest to find out whetherthese tumors form a clinical subgroup. Finally, chromosometranslocations involving region q13-g15 of human chromosome 12 have beenreported for a variety of other solid tumors: benign adipose tissuetumors, uterine leiomyoma, rhabdomyosarcoma, hemangiopericytoma,clear-cell sarcoma, chondromatous tumors, and hamartoma of the lung.Whether or not the chromosome 12 breakpoints in some of these tumors mapwithin the same region as that of Ad-312/SV40 remains to be established.The YAC and cosmid clones described in this report constitute usefultools to investigate this.

[0434] The availability of a copy of the first-generation CEPH YAClibrary [10] and a copy of the arrayed chromosome 12-specific cosmidlibrary (LLNL12NC01) [11] is greatly acknowledged. The cosmid librarywas constructed as part of the National. Laboratory Gene Library Projectunder the auspices of the U.S. DOE by LLNL under contract No.W-7405-Eng-48. The authors acknowledge the excellent technicalassistance of M. Dehaen, C. Huysmans, E. Meyen, K. Meyer-Bolte, and M.Willems and would like to thank M. Leys for art-work. This work wassupported in part by the EC through Biomed 1 program “MolecularCytogenetics of Solid Tumours”, the “Geconcerteerde Onderzoekacties1992-1996”, the “Association Luxembourgeoise contre le Cancer”, theNational Fund for Scientific Research (NFWO; Kom op tegen Kanker), the“ASLK-programma voor Kankeronderzoek”, the “Schwerpunktprogramm:Molekulare und Klassische Tumorcytogenetik” of the DeutscheForschungsgemeinschaft, and the Tönjes-Vagt Stiftung. This text presentsresults of the Belgian programme on Interuniversity Poles of Attractioninitiated by the Belgian State, Prime Minister's Office, Science PolicyProgramming. The scientific responsibility is assumed by its authors. J.W. M. Geurts is an “Aspirant” of the National Fund for ScientificResearch (NFWO; Kom op tegen Kanker).

REFERENCES

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[0436] 2. Sandros J, Stenman G, Mark J (1990): Cytogenetic and molecularobservations in human and experimental salivary gland tumours. CancerGenet Cytogenet 44: 153-167.

[0437] 3. Bullerdiek J, Wobst G, Meyer-Bolte K, Chilla R, Haubrich J,Thode B, Bartnitzke S (1993): Cytogenetic subtyping of 220 salivarygland pleomorphic adenomas: correlation to occurrence, histologicalsubtype, and in vitro cellular behavior. Cancer Genet Cytogenet 65:27-31.

[0438] 4. Schoenmakers H F P M, Kools P F J, Kazmierczak B, BullerdiekJ, Claussen U, Horsthemke B, Van den Berghe H Van de Ven W J M (1993):Isolation of a somatic cell hybrid retaining the der(16)t(12;16)(q13;p11.2) from a myxoid liposarcoma cell line. Cell Genet Cytogenet62: 159-161.

[0439] 5. Schoenmakers H F P M, Kools P F J, Mols R, Kazmierczak B,Bartnitzke S,. Bullerdiek J, Dal Cin P, De Jong P J, Van den Berghe H,Van de Ven W J M (1993): Physical mapping of chromosome 12q breakpointsin lipoma, pleomorphic salivary gland adenoma, uterine leiomyoma, andmyxoid liposarcoma. Genomics, 20: 210-222.

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[0457] Legends of Figures of ANNEX 2

[0458]FIG. 1. Composite physical map of the overlapping DNA inserts ofYAC clones Y4854 and Y9091. Sizes of the DNA inserts are indicated. Therelative positions of the YAC clones are represented by bars below thelong range physical map. Sequence-tagged sites (STSs) corresponding toend-clones of YACs, including YACs not shown here, are indicated byboxed RM codes above the restriction map. STSs obtained frominter-Alu-PCR products are given below the restriction map and the DNAregions to which they have been mapped are marked by arrows. B: BssHII;M: MluI; P: PvuI; Sf: SfiI. A polymorphic MluI site is marked by anasterisk.

[0459]FIG. 2. A) Mapping of cosmid clone cPK12qter to the telomericregion of the long arm of chromosome 12. Centromere 12-specific probepα12H8 was used to establish the identity of chromosome 12. FISHanalysis was performed on metaphase chromosomes of control humanlymphocytes. Hybridization signals of cPK12qter are marked with smallarrowheads, those of the centromere 12-specific probe with asterisks. B,C) FISH analysis of metaphase chromosomes of Ad-312/SV40 cells using DNAof YAC clone Y4854 (B) or Y9091 (C) as molecular probe in combinationwith cosmid clone cPK12qter as reference marker. Hybridization signalsof the YAC clones on chromosome 12 are indicated by large arrowheads;those on der(1) by large arrows, and those on der(12) by small arrows,respectively. The hybridization signals of cosmid clone cPK12qter areindicated by small arrowheads.

[0460]FIG. 3. FISH analysis of metaphase chromosomes of Ad-312/SV40cells using DNA of cosmid clone cRM69 (A) or cRM111 (B) as molecularprobe in combination with cosmid clone cPK12qter as reference marker.The position of the hybridization signals of cPK12qter are indicated bysmall arrowheads. In (A), the position of the hybridization signal ofcRM69 on normal chromosome 12 is indicated by a large arrowhead, andthat on der(12) with a small arrow. In (B), the position of thehybridization signal of cRM111 on normal chromosome 12 is indicated by alarge arrowhead, and that on der(1) with a large arrow.

[0461]FIG. 4. Schematic representation of FISH mapping data obtained forthe six pleomorphic salivary gland adenoma cell lines tested in thisstudy. The specific chromosome 12 aberrations in the various cell linesare given. Cosmid clones which were used as probes in the FISH mappingstudies correspond to sequence-tagged sites obtained from overlappingYAC clones. Individual FISH experiments are indicated by dots. Cosmidclones were named after the acronyms of the STSs, as shown in the boxes,and the relative order of these is as presented. The DNA intervalbetween RM90 and RM103 is estimated to be about 1.3 Mb. Insert:Schematic representation of the G-banded derivative chromosomes der(1)and der(12) of the Ad-312/SV40 cell line, which carries a t(1;12)(p22;q15). The positions of the chromosome 12 breakpoints ofAd-248/SV40, Ad-263/SV40, Ad-295/SV40, Ad-302/SV40, and Ad-366/SV40 aredistal to RM103 as indicated by the arrow.

1 164 34 base pairs nucleic acid single linear Other 1 AAGGATCCGTCGACATCTTT TTTTTTTTTT TTTT 34 29 base pairs nucleic acid single linearOther 2 CUACUACUAC UAAAGGATCC GTCGACATC 29 18 base pairs nucleic acidsingle linear Other 3 CTTCAGCCCA GGGACAAC 18 18 base pairs nucleic acidsingle linear Other 4 CAAGAGGCAG ACCTAGGA 18 23 base pairs nucleic acidsingle linear Other 5 AACAATGCAA CTTTTAATTA CTG 23 30 base pairs nucleicacid single linear Other 6 CAUCAUCAUC AUCGCCTCAG AAGAGAGGAC 30 30 basepairs nucleic acid single linear Other 7 CAUCAUCAUC AUGTTCAGAAGAAGCCTGCT 30 35 base pairs nucleic acid single linear Other 8CAUCAUCAUC AUTTGATCTG ATAAGCAAGA GTGGG 35 21 base pairs nucleic acidsingle linear 9 CTCCAAGACA GGCCTCTGAT G 21 22 base pairs nucleic acidsingle linear 10 ACCACAGGTC CCCTTCAAAC TA 22 18 base pairs nucleic acidsingle linear 11 TCCTCCTGAG CAGGCTTC 18 18 base pairs nucleic acidsingle linear 12 CTTCAGCCCA GGGACAAC 18 18 base pairs nucleic acidsingle linear 13 CGCCTCAGAA GAGAGGAC 18 25 amino acids amino acid singlelinear None 14 Ala Arg Gly Glu Gly Ala Gly Gln Pro Ser Thr Ser Ala GlnGly Gln 1 5 10 15 Pro Ala Ala Pro Ala Pro Gln Lys Arg 20 25 17 aminoacids amino acid single linear None 15 Ser Pro Ser Lys Ala Ala Gln LysLys Ala Glu Ala Thr Gly Glu Lys 1 5 10 15 Arg 17 amino acids amino acidsingle linear None 16 Pro Arg Lys Trp Pro Gln Gln Val Val Gln Lys LysPro Ala Gln Glu 1 5 10 15 Glu 20 base pairs nucleic acid single linear17 TGGGACTAAC GGATTTTCAA 20 20 base pairs nucleic acid single linear 18TGTGGTTCAT TCATGCATTA 20 20 base pairs nucleic acid single linear 19TCCATCATCA TCTCAAAACA 20 22 base pairs nucleic acid single linear 20CTCTACCAAA TGGAATAAAC AG 22 20 base pairs nucleic acid single linear 21GCAGCTCAGG CTCCTTCCCA 20 20 base pairs nucleic acid single linear 22TGGCTTCCTG AAACGCGAGA 20 20 base pairs nucleic acid single linear 23TCTCCACTGC TTCCATTCAC 20 20 base pairs nucleic acid single linear 24ACACAAAACC ACTGGGGTCT 20 20 base pairs nucleic acid single linear 25CAGCTTTGGA ATCAGTGAGG 20 20 base pairs nucleic acid single linear 26CCTGGGGAAG AGGAGTAAAG 20 18 base pairs nucleic acid single linear 27GAGCTTCCTA TCTCATCC 18 18 base pairs nucleic acid single linear 28ATGCTTGTGT GTGAGTGG 18 18 base pairs nucleic acid single linear 29TTTGCTAAGC TAGGTGCC 18 18 base pairs nucleic acid single linear 30AGCTTCAAGA CCCATGAG 18 18 base pairs nucleic acid single linear 31CAGTTCTGAG ACTGCTTG 18 18 base pairs nucleic acid single linear 32TAATAGCAGG GACTCAGC 18 22 base pairs nucleic acid single linear 33CTTGTCTCAT TCTTTTAAAG GG 22 20 base pairs nucleic acid single linear 34CACCCCTTTT TAGATCCTAC 20 20 base pairs nucleic acid single linear 35GAATGTTCAT CACAGTGCTG 20 20 base pairs nucleic acid single linear 36AATGTGAGGT TCTGCTGAAG 20 20 base pairs nucleic acid single linear 37TTCTCATGGG GTAAGGACAG 20 22 base pairs nucleic acid single linear 38AAAGCTGCTT ATATAGGGAA TC 22 22 base pairs nucleic acid single linear 39CCTTGGCTTA GATATGATAC AC 22 22 base pairs nucleic acid single linear 40GCTCTTCAGA AATATCCTAT GG 22 20 base pairs nucleic acid single linear 41CCTTAGCAGT TGCTTGTCTG 20 20 base pairs nucleic acid single linear 42TCGTCACAGG ACATAGTCAC 20 23 base pairs nucleic acid single linear 43TCTATGGTAT GTTATACAAG ATG 23 19 base pairs nucleic acid single linear 44CAGTGAGATC CTGTCTCTA 19 24 base pairs nucleic acid single linear 45TCTGTGATGT TTTAAGCCAC TTAG 24 20 base pairs nucleic acid single linear46 AATTCTGTGT CCCTGCCACC 20 20 base pairs nucleic acid single linear 47ATTCTTCCTC ACCTCCCACC 20 20 base pairs nucleic acid single linear 48AATCTGCAGA GAGGTCCAGC 20 20 base pairs nucleic acid single linear 49AATTCTCCAT CTGGGCCTGG 20 20 base pairs nucleic acid single linear 50GAACGCTAAG CATGTGGGAG 20 20 base pairs nucleic acid single linear 51CTCCAACCAT GGTCCAAAAC 20 20 base pairs nucleic acid single linear 52GACCTCCAGT GGCTCTTTAG 20 20 base pairs nucleic acid single linear 53ACCATCAGAT CTGGCACTGA 20 20 base pairs nucleic acid single linear 54TTACATTGGA GCTGTCATGC 20 20 base pairs nucleic acid single linear 55TCCAGGACAT CCTGAAAATG 20 20 base pairs nucleic acid single linear 56AGTATCCTGC ACTTCTGCAG 20 21 base pairs nucleic acid single linear 57GATGAACTCT GAGGTGCCTT C 21 20 base pairs nucleic acid single linear 58TCAAACCCAG CTTTGACTCC 20 20 base pairs nucleic acid single linear 59GTCTTCAAAA CGCTTTCCTG 20 20 base pairs nucleic acid single linear 60TGGTTTGCAT AATGGTGATG 20 20 base pairs nucleic acid single linear 61TACACTACTC TGCAGCACAC 20 20 base pairs nucleic acid single linear 62TCTGAGTCAA TCACATGTCC 20 20 base pairs nucleic acid single linear 63CTCCCCAGAT GATCTCTTTC 20 20 base pairs nucleic acid single linear 64CGGTAGGAAA TAAAGGAGAG 20 20 base pairs nucleic acid single linear 65TATTTACTAG CTGGCCTTGG 20 20 base pairs nucleic acid single linear 66CATCTCAGGC ACACACAATG 20 20 base pairs nucleic acid single linear 67ATTCAGAGAA GTGGCCAAGT 20 20 base pairs nucleic acid single linear 68GGGATAGGTC TTCTGCAATC 20 20 base pairs nucleic acid single linear 69TCCAACAATA CTGAGTGACC 20 20 base pairs nucleic acid single linear 70TCCATTTCAC TGTAGCACTG 20 20 base pairs nucleic acid single linear 71GTAATCAACC ATTCCCCTGA 20 20 base pairs nucleic acid single linear 72AAAATAGCTG GTATGGTGGC 20 20 base pairs nucleic acid single linear 73ACTGCTCTAG TTTTCAAGGA 20 20 base pairs nucleic acid single linear 74AATTTACCTG ACAGTTTCCT 20 20 base pairs nucleic acid single linear 75GCATTTGACG TCCAATATTG 20 20 base pairs nucleic acid single linear 76ATTCCATTGG CTAACACAAG 20 20 base pairs nucleic acid single linear 77GCAAAACTTT GACTGAAACG 20 20 base pairs nucleic acid single linear 78CACAGAGTAT CGCACTGCAT 20 20 base pairs nucleic acid single linear 79AAGAGATTTC CCATGTTGTG 20 20 base pairs nucleic acid single linear 80CTAGTGCCTT CACAAGAACC 20 20 base pairs nucleic acid single linear 81AATTCTTGAG GGGTTCACTG 20 20 base pairs nucleic acid single linear 82TCCACACTGA GAGCTTTTCA 20 20 base pairs nucleic acid single linear 83GTGGTTCTGT ACAGCAGTGG 20 20 base pairs nucleic acid single linear 84TGAGAAAATG TCTGCCAAAT 20 20 base pairs nucleic acid single linear 85GCTCTACCAG GCATACAGTG 20 20 base pairs nucleic acid single linear 86ATTCCTAGCA TCTTTTCACG 20 20 base pairs nucleic acid single linear 87ATATGCATTA GGCTCAACCC 20 20 base pairs nucleic acid single linear 88ATCCCACAGG TCAACATGAC 20 25 base pairs nucleic acid single linear 89ATCCTTACAT TTCCAGTGGC ATTCA 25 23 base pairs nucleic acid single linear90 CCCAGAAGAC CCACATTCCT CAT 23 25 base pairs nucleic acid single linear91 TTTTAAGTTT CTCCAGGGAG GAGAC 25 25 base pairs nucleic acid singlelinear 92 AATAGGCTCT TTGGAAAGCT GGAGT 25 26 base pairs nucleic acidsingle linear 93 TCTCAGCTTA ATCCAAGAAG GACTTC 26 26 base pairs nucleicacid single linear 94 GGCATATTCC TCAACAATTT ATGCTT 26 25 base pairsnucleic acid single linear 95 TGGAGAAGCT ATGGTGCTTC CTATG 25 28 basepairs nucleic acid single linear 96 TGACAAATAG GTGAGGGAAA GTTGTTAT 28 20base pairs nucleic acid single linear 97 TCACACGCTG AATCAATCTT 20 20base pairs nucleic acid single linear 98 CAGCAGCTGA TACAAGCTTT 20 20base pairs nucleic acid single linear 99 TGTTTTCTTT CCCGATAGGT 20 20base pairs nucleic acid single linear 100 CTGGGATGCT CTTCGACCTC 20 22base pairs nucleic acid single linear 101 CCATCCAACA TCTTAAATGG AC 22 23base pairs nucleic acid single linear 102 CAGCTGCAAA CTCTAGGACT ATT 2310 base pairs nucleic acid single linear 103 TAGGAAATGG 10 10 base pairsnucleic acid single linear 104 GTGAGTAATA 10 10 base pairs nucleic acidsingle linear 105 AATACTCTGG 10 10 base pairs nucleic acid single linear106 CCTACTATTG 10 10 base pairs nucleic acid single linear 107GGAAGTGTGA 10 10 base pairs nucleic acid single linear 108 AACACAGGAC 1010 base pairs nucleic acid single linear 109 GTTTAATATT 10 10 base pairsnucleic acid single linear 110 AAGAAGGCAG 10 10 base pairs nucleic acidsingle linear 111 TAGGAGGTAG 10 10 base pairs nucleic acid single linear112 GGTGGCCATT 10 10 base pairs nucleic acid single linear 113GACAATCTAC 10 10 base pairs nucleic acid single linear 114 GTACAGAAGA 1010 base pairs nucleic acid single linear 115 GGGCATTCAG 10 10 base pairsnucleic acid single linear 116 GCAGTCTGTA 10 10 base pairs nucleic acidsingle linear 117 TCTGTATCCT 10 10 base pairs nucleic acid single linear118 ACACACTTCC 10 10 base pairs nucleic acid single linear 119ATATTATGGA 10 10 base pairs nucleic acid single linear 120 GAGGAGTTTT 1010 base pairs nucleic acid single linear 121 TAACACAGGA 10 10 base pairsnucleic acid single linear 122 TTACCTGCTG 10 10 base pairs nucleic acidsingle linear 123 GCTGGAGTGC 10 10 base pairs nucleic acid single linear124 GTCTCCTCCC 10 10 base pairs nucleic acid single linear 125TTTNTCTCTT 10 10 base pairs nucleic acid single linear 126 AGTCCAAGAA 1010 base pairs nucleic acid single linear 127 CCAAACTCTG 10 10 base pairsnucleic acid single linear 128 CTCCAGAAAC 10 10 base pairs nucleic acidsingle linear 129 AACTTCTTGA 10 10 base pairs nucleic acid single linear130 GAATGTCAGA 10 10 base pairs nucleic acid single linear 131CCTGGAAGCT 10 10 base pairs nucleic acid single linear 132 ATGGAGTCTC 1010 base pairs nucleic acid single linear 133 TTCCAGATAC 10 10 base pairsnucleic acid single linear 134 GCCTGCTCAG 10 10 base pairs nucleic acidsingle linear 135 TATCCTTTCA 10 10 base pairs nucleic acid single linear136 TCTTTCCACT 10 10 base pairs nucleic acid single linear 137ATACCACTTA 10 10 base pairs nucleic acid single linear 138 TTGCCATGGT 1010 base pairs nucleic acid single linear 139 CACTTTCATC 10 10 base pairsnucleic acid single linear 140 ATAAGGACTA 10 10 base pairs nucleic acidsingle linear 141 NCTTGTNAGC 10 10 base pairs nucleic acid single linear142 GTAAGACATA 10 10 base pairs nucleic acid single linear 143GTCAATGTTG 10 10 base pairs nucleic acid single linear 144 TCCTGGTACC 1010 base pairs nucleic acid single linear 145 TCTTTCAGAT 10 10 base pairsnucleic acid single linear 146 AATTACCTCT 10 10 base pairs nucleic acidsingle linear 147 GACTGACTCA 10 10 base pairs nucleic acid single linear148 TATTCCTGAA 10 10 base pairs nucleic acid single linear 149GTCAATGTTG 10 10 base pairs nucleic acid single linear 150 AATTACCTCT 1010 base pairs nucleic acid single linear 151 GCTTTTTCAA 10 10 base pairsnucleic acid single linear 152 GTTAAGAAAC 10 10 base pairs nucleic acidsingle linear 153 GNCTGACTAC 10 10 base pairs nucleic acid single linear154 AAGTCAAGAG 10 10 base pairs nucleic acid single linear 155TTTTAAAACA 10 10 base pairs nucleic acid single linear 156 AATCTGAAAT 1010 base pairs nucleic acid single linear 157 ATATGGCAAG 10 10 base pairsnucleic acid single linear 158 TCAGGCATCA 10 10 base pairs nucleic acidsingle linear 159 TAGAGATTAG 10 1033 base pairs nucleic acid singlelinear cDNA 160 CGCTTCAGAA GAGAGGACGC GGCCGCCCCA GGAAGCAGCA GCAAAAACCAACCGGTGAGC 60 CCTCTCCTAA GAGACCCAGG GGAAGACCCA AAGGCAGCAA AAACAAGAGTCCCTCTAAAG 120 CAGCTCAAGA GGAAGCAGAA GCCACTGAAG AAAAACGGCC AAGGGGCAGACCTAGGAAAT 180 GGGGTGGCCA TTCAGGGCAA CTGGGGCCTT CGTCAGTTGC CCCTTCATTCCGCCCAGAGG 240 ATGAGCTTGA GCACCTGACC AAAAAGATGC TGTATGACAT GGAAAATCCACCTGCTGACG 300 AATACTTTGG CCGCTGTGCT CGCTGTGGAG AAAACGTAGT TGGGGAAGGTACAGGATGCA 360 CTGCCATGGA TCAGGTCTTC CACGTGGATT GTTTTACCTG CATCATCTGCAACAACAAGC 420 TCCGAGGGCA GCCATTCTAT GCTGTGGAAA AGAAAGCATA CTGCGAGCCCTGCTACATTA 480 ATACTCTGGA GCAGTGCAAT GTGTGTTCCA AGCCCATCAT GGAGCGGATTCTCCGAGCCA 540 CCGGGAAGGC CTATCATCCT CACTGTTTCA CCTGCGTGAT GTGCCACCGCAGCCTGGATG 600 GGATCCCATT CACTGTGGAT GCTGGCGGGC TCATTCACTG CATTGAGGACTTCCACAAGA 660 AATTTGCCCC GCGGTGTTCT GTGTGCAAGG AGCCTATTAT GCCAGCCCCGGGCCAGGAGG 720 AGACTGTCCG TATTGTGGCT TTGGATCGAG ATTTCCATGT TCACTGCTACCGATGCGAGG 780 ATTGCGGTGG TCTCCTGTCT GAAGGAGATA ACCAAGGCTG CTACCCCTTGGATGGGCACA 840 TCCTCTGCAA GACCTGCAAC TCTGCCCGCA TCAGGGTGTT GACCGCCAAGGCGAGCACTG 900 ACCTTTAGAT TCAGTCACCT GTTCAGCCGG CACTGAGAAG AACGAACACAAGAAAAAGAT 960 AAGAAATACT AGAGTAAAGG CCATCAAACT ACGCGAAAAA AAAAAAAAAAAAAAAAGATG 1020 TCGACGGATC CTT 1033 343 amino acids amino acid singlelinear None 161 Leu Gln Lys Arg Gly Arg Gly Arg Pro Arg Lys Gln Gln GlnLys Pro 1 5 10 15 Thr Gly Glu Pro Ser Pro Lys Arg Pro Arg Gly Arg ProLys Gly Ser 20 25 30 Lys Asn Lys Ser Pro Ser Lys Ala Ala Gln Glu Glu AlaGlu Ala Thr 35 40 45 Glu Glu Lys Arg Pro Arg Gly Arg Pro Arg Lys Trp GlyGly His Ser 50 55 60 Gly Gln Leu Gly Pro Ser Ser Val Ala Pro Ser Phe ArgPro Glu Asp 65 70 75 80 Glu Leu Glu His Leu Thr Lys Lys Met Leu Tyr AspMet Glu Asn Pro 85 90 95 Pro Ala Asp Glu Tyr Phe Gly Arg Cys Ala Arg CysGly Glu Asn Val 100 105 110 Val Gly Glu Gly Thr Gly Cys Thr Ala Met AspGln Val Phe His Val 115 120 125 Asp Cys Phe Thr Cys Ile Ile Cys Asn AsnLys Leu Arg Gly Gln Pro 130 135 140 Phe Tyr Ala Val Glu Lys Lys Ala TyrCys Glu Pro Cys Tyr Ile Asn 145 150 155 160 Thr Leu Glu Gln Cys Asn ValCys Ser Lys Pro Ile Met Glu Arg Ile 165 170 175 Leu Arg Ala Thr Gly LysAla Tyr His Pro His Cys Phe Thr Cys Val 180 185 190 Met Cys His Arg SerLeu Asp Gly Ile Pro Phe Thr Val Asp Ala Gly 195 200 205 Gly Leu Ile HisCys Ile Glu Asp Phe His Lys Lys Phe Ala Pro Arg 210 215 220 Cys Ser ValCys Lys Glu Pro Ile Met Pro Ala Pro Gly Gln Glu Glu 225 230 235 240 ThrVal Arg Ile Val Ala Leu Asp Arg Asp Phe His Val His Cys Tyr 245 250 255Arg Cys Glu Asp Cys Gly Gly Leu Leu Ser Glu Gly Asp Asn Gln Gly 260 265270 Cys Tyr Pro Leu Asp Gly His Ile Leu Cys Lys Thr Cys Asn Ser Ala 275280 285 Arg Ile Arg Val Leu Thr Ala Lys Ala Ser Thr Asp Leu Xaa Ile Gln290 295 300 Ser Pro Val Gln Pro Ala Leu Arg Arg Thr Asn Thr Arg Lys ArgXaa 305 310 315 320 Glu Ile Leu Glu Xaa Arg Pro Ser Asn Tyr Ala Lys LysLys Lys Lys 325 330 335 Lys Lys Asp Val Asp Gly Ser 340 4323 base pairsnucleic acid single linear cDNA 162 GTCACTTTTA TTTGGGGGTG TGGACAGCTGCTTTCCCAGG GGAGTACTTC TTACAGTGGG 60 ATTTCAAGAC AAGATCGGCC TGAAGAAAAATTATATTTGT ATATTTTTTA AAAAGTGGGA 120 ACTTTGAGGC TCAGAGACAG AGCAGAAGACAGAACCTGGT CTTCTGATTC CCTGTGTTCT 180 GCTTTTTTCA TTGTTCCACT GGACGCTCATCAGAGGGAAG ATCTTTTTCC TCAATTGATT 240 CCAACAATGT CTCACCCATC TTGGCTGCCACCCAAAAGCA CTGGTGAGCC CCTCGGCCAT 300 GTGCCTGCAC GGATGGAGAC CACCCATTCCTTTGGGAACC CCAGCATTTC AGTGTCTACA 360 CAACAGCCAC CCAAAAAGTT TGCCCCGGTAGTTGCTCCAA AACCTAAGTA CAACCCATAC 420 AAACAACCTG GAGGTGAGGG TGATTTTCTTCCACCCCCAC CTCCACCTCT AGATGATTCC 480 AGTGCCCTTC CATCTATCTC TGGAAACTTTCCTCCTCCAC CACCTCTTGA TGAAGAGGCT 540 TTCAAAGTAC AGGGGAATCC CGGAGGCAAGACACTTGAGG AGAGGCGCTC CAGCCTGGAC 600 GCTGAGATTG ACTCCTTGAC CAGCATCTTGGCTGACCTTG AGTGCAGCTC CCCCTATAAG 660 CCTCGGCCTC CACAGAGCTC CACTGGTTCAACAGCCTCTC CTCCAGTTTC GACCCCAGTC 720 ACAGGACACA AGAGAATGGT CATCCCGAACCAACCCCCTC TAACAGCAAC CAAGAAGTCT 780 ACATTGAAAC CACAGCCTGC ACCCCAGGCTGGACCCATCC CTGTGGCTCC AATCGGAACA 840 CTCAAACCCC AGCCTCAGCC AGTCCCAGCCTCCTACACCA CGGCCTCCAC TTCTTCAAGG 900 CCTACCTTTA ATGTGCAGGT GAAGTCAGCCCAGCCCAGCC CTCATTATAT GGCTGCCCCT 960 TCATCAGGAC AAATTTATGG CTCAGGGCCCCAGGGCTATA ACACTCAGCC AGTTCCTGTC 1020 TCTGGGCAGT GTCCACCTCC TTCAACACGGGGAGGCATGG ATTATGCCTA CATTCCACCA 1080 CCAGGACTTC AGCCGGAGCC TGGGTATGGGTATGCCCCCA ACCAGGGACG CTATTATGAA 1140 GGCTACTATG CAGCAGGGCC AGGCTATGGGGGCAGAAATG ACTCTGACCC TACCTATGGT 1200 CAACAAGGTC ACCCAAATAC CTGGAAACGGGAACCAGGGT ACACTCCTCC TGGAGCAGGG 1260 AACCAGAACC CTCCTGGGAT GTATCCAGTCACTGGTCCCA AGAAGACCTA TATCACAGAT 1320 CCTGTTTCAG CCCCCTGTGC GCCACCATTGCAGCCAAAGG GTGGCCATTC AGGGCAACTG 1380 GGGCCTTCGT CAGTTGCCCC TTCATTCCGCCCAGAGGATG AGCTTGAGCA CCTGACCAAA 1440 AAGATGCTGT ATGACATGGA AAATCCACCTGCTGACGAAT ACTTTGGCCG CTGTGCTCGC 1500 TGTGGAGAAA ACGTAGTTGG GGAAGGTACAGGATGCACTG CCATGGATCA GGTCTTCCAC 1560 GTGGATTGTT TTACCTGCAT CATCTGCAACAACAAGCTCC GAGGGCAGCC ATTCTATGCT 1620 GTGGAAAAGA AAGCATACTG CGAGCCCTGCTACATTAATA CTCTGGAGCA GTGCAATGTG 1680 TGTTCCAAGC CCATCATGGA GCGGATTCTCCGAGCCACCG GGAAGGCCTA TCATCCTCAC 1740 TGTTTCACCT GCGTGATGTG CCACCGCAGCCTGGATGGGA TCCCATTCAC TGTGGATGCT 1800 GGCGGGCTCA TTCACTGCAT TGAGGACTTCCACAAGAAAT TTGCCCCGCG GTGTTCTGTG 1860 TGCAAGGAGC CTATTATGCC AGCCCCGGGCCAGGAGGAGA CTGTCCGTAT TGTGGCTTTG 1920 GATCGAGATT TCCATGTTCA CTGCTACCGATGCGAGGATT GCGGTGGTCT CCTGTCTGAA 1980 GGAGATAACC AAGGCTGCTA CCCCTTGGATGGGCACATCC TCTGCAAGAC CTGCAACTCT 2040 GCCCGCATCA GGGTGTTGAC CGCCAAGGCGAGCACTGACC TTTAGATTCA GTCACCTGTT 2100 CAGCCGGCAC TGAGAAGAAC GAACACAAGAAAAAGATAAG AAATACTAGA GTAAAGGCCA 2160 TCAAACTACG CGATAGTCTC TGTTCTTCATCTGCTATTAA CCTTGCCTTA GAAACACATA 2220 AATTATGAGA TTTTTTTTTA AAAGTTGTTACCAAATACAC ATTTCACATT GAATCATGTA 2280 GGATCTTGAT GGGCCTTTGT TCCCAAGGACTTCCACATTT TTGCACAGAT TATGCTCCAT 2340 CCCTTCACTT CTGCATTCCT GTAACTTTTAATCCCTATGT TTGTCTCACT TTTCATCTGG 2400 TTGAATGGCT TTTCTTAGTG TGGTATTTGCTGTCACATAG TTTTTTCCTG GGTGAGTCTG 2460 CCAACTCACA GGTGCTTTTA GGCTTGAAATCTCCATCCTA TCATTTCCGT TTTGCCTGTG 2520 ACTGTAAAGA GTAGCCATTC TTTTCCCATGTATTGAAGAG GATATTCTTC TCTTGCTTTA 2580 TACTACTCAC GTCCTTGGGG AGGGAAATGCACAATTTTTT TTTGTTAGGC TGTAAAGAAT 2640 TTAAGCTGTA AATTACATAA GTTAGAACAAGCCCAAATTT AATTTGCAAC CATCAGAATT 2700 CAGAATCTAT AGTGACCAGT GATCAAGGCTAATTGGAAAA GAGTTATCGG CCCATAGCTA 2760 ATAAGTAGTG ACAGACAACC AAGCTTCAATATTTTTCTAA AGAAATTACA GGTGGGATAT 2820 GCTAGAAAAG GCATTTTGGG GTTATGTTTAAAAAAACATT ATTGTCCCAC AATATTACCT 2880 TAAGATTTTT CTTTTCCGCA CTACCTGAACATTGTAATAC AGACAAACTT GATTTCTTCT 2940 AGAAGATAAC ATTTTCAATA CTGTCCCACTTCTCATCTTA AAAATATTGT CATGTTTATT 3000 CTAATATCCA ACGCAACTAT CAAAATTGCCTTTTTCTCTA GAGGATGAAG GCTGTGAAAA 3060 AACCGTTCAA ATTCTCTTCT TTTTCTTTTTTATTACCAGG TCCATTTTGC CTGACAATTG 3120 CAAATCAGAG CATACAAAAT AAAACTGTGCAGTTTTGTTT GGTTTACTTT CAAAAGAGTA 3180 GAAAGCTTGA AAAGATTCTG AAACCACAGTTTCATTATTC TCATAATCCT TCTGCAACTG 3240 AAATTACATA TTGCAGGAGA CATTTTCATATCATCAATCT GACATTTACA CCACACTTTC 3300 AAAGACAATC ACTGAAACAA AAATTGTCTTTATGAGCTAA AAATATGCAG AATCTCTGCC 3360 TAGAATCTTT ATTCAAACTT TTATTAGCCAGTGAAACACT TGCTTGCCAA CTGCCAAGCC 3420 ATACTTATTA AGTTCGAACA TGTTTCACTTAAGGAGAGAC ACCTAGCTTA GTCATGGCAA 3480 GTTGCCATTT TGTAAACTAA GGATTTTGGACTGAGATTTC TTAAATCTTT CTTCAAATCT 3540 CCCACAAGTA TATACTTTTA AATTATGGAGTATTTTAAGT CTACAAAAAG GTATAAATAA 3600 TAATATAATG AATTCCTATA TACCTAATACCCAGTTTAAG ACACCAAATA TAACAAGTAT 3660 AATTACATCC TCCAATGTAC CGTTTCCTTATTCCACAGAT ATCTTTTTCA TTATTGTGAA 3720 GTGATGTTCA GATTTCTAGT TTTTTTTTCTAGTTTTTAAT TTTAACATCA GAACTGAAAT 3780 AAAAAATTAT GGATACGTGT TTTGAATTGCAAACTATTCC TCAGGAATTC CAATTAAATT 3840 TATTTTACTT GAATAGGAAT GATCATAAAAGTGATTCTTT TTTTGTGACT AGAAATTCTT 3900 AAGCCGATGG TCACTATAGC TCATCCTTAATGTATGGCTC ATTTGCTTTT GTCACTAAAC 3960 GGTTTTGTGT TAGAACCACC AAAATTATAGCTTTTAAGAG CTTCCTTTGA CCACTGTCTT 4020 TTTCTTACCC TACTTCTCTT ATCTTTGATCGTATATTTCT CATAATGTGA AATATGATGA 4080 GATTCACTTA GGGGCAGCAT GTTAGTTTTGGGAGGCAATG TCAACTGTGT CTCTGAATTC 4140 CTGTCTTCCA AATTGAAGCC AGACCATGCTGATGACCTCA AGTAGCACTG ACTATTTGAC 4200 AATAGGGCTG ATAATGTAAT CGGCTTGAATTTTGACTTAG TAACTTTTTA TGTAATACTT 4260 TCGGAGAAAT TCTCTTTAGG ACAAAGCAGAGAGTCCAATT TATTGAGGGA TAGATTGTAT 4320 CTC 4323 612 amino acids aminoacid single linear 163 Met Ser His Pro Ser Trp Leu Pro Pro Lys Ser ThrGly Glu Pro Leu 1 5 10 15 Gly His Val Pro Ala Arg Met Glu Thr Thr HisSer Phe Gly Asn Pro 20 25 30 Ser Ile Ser Val Ser Thr Gln Gln Pro Pro LysLys Phe Ala Pro Val 35 40 45 Val Ala Pro Lys Pro Lys Tyr Asn Pro Tyr LysGln Pro Gly Gly Glu 50 55 60 Gly Asp Phe Leu Pro Pro Pro Pro Pro Pro LeuAsp Asp Ser Ser Ala 65 70 75 80 Leu Pro Ser Ile Ser Gly Asn Phe Pro ProPro Pro Pro Leu Asp Glu 85 90 95 Glu Ala Phe Lys Val Gln Gly Asn Pro GlyGly Lys Thr Leu Glu Glu 100 105 110 Arg Arg Ser Ser Leu Asp Ala Glu IleAsp Ser Leu Thr Ser Ile Leu 115 120 125 Ala Asp Leu Glu Cys Ser Ser ProTyr Lys Pro Arg Pro Pro Gln Ser 130 135 140 Ser Thr Gly Ser Thr Ala SerPro Pro Val Ser Thr Pro Val Thr Gly 145 150 155 160 His Lys Arg Met ValIle Pro Asn Gln Pro Pro Leu Thr Ala Thr Lys 165 170 175 Lys Ser Thr LeuLys Pro Gln Pro Ala Pro Gln Ala Gly Pro Ile Pro 180 185 190 Val Ala ProIle Gly Thr Leu Lys Pro Gln Pro Gln Pro Val Pro Ala 195 200 205 Ser TyrThr Thr Ala Ser Thr Ser Ser Arg Pro Thr Phe Asn Val Gln 210 215 220 ValLys Ser Ala Gln Pro Ser Pro His Tyr Met Ala Ala Pro Ser Ser 225 230 235240 Gly Gln Ile Tyr Gly Ser Gly Pro Gln Gly Tyr Asn Thr Gln Pro Val 245250 255 Pro Val Ser Gly Gln Cys Pro Pro Pro Ser Thr Arg Gly Gly Met Asp260 265 270 Tyr Ala Tyr Ile Pro Pro Pro Gly Leu Gln Pro Glu Pro Gly TyrGly 275 280 285 Tyr Ala Pro Asn Gln Gly Arg Tyr Tyr Glu Gly Tyr Tyr AlaAla Gly 290 295 300 Pro Gly Tyr Gly Gly Arg Asn Asp Ser Asp Pro Thr TyrGly Gln Gln 305 310 315 320 Gly His Pro Asn Thr Trp Lys Arg Glu Pro GlyTyr Thr Pro Pro Gly 325 330 335 Ala Gly Asn Gln Asn Pro Pro Gly Met TyrPro Val Thr Gly Pro Lys 340 345 350 Lys Thr Tyr Thr Thr Asp Pro Val SerAla Pro Cys Ala Pro Pro Leu 355 360 365 Gln Pro Lys Gly Gly His Ser GlyGln Leu Gly Pro Ser Ser Val Ala 370 375 380 Pro Ser Phe Arg Pro Glu AspGlu Leu Glu His Leu Thr Lys Lys Met 385 390 395 400 Leu Tyr Asp Met GluAsn Pro Pro Ala Asp Glu Tyr Phe Gly Arg Cys 405 410 415 Ala Arg Cys GlyGlu Asn Val Val Gly Glu Gly Thr Gly Cys Thr Ala 420 425 430 Met Asp GlnVal Phe His Val Asp Cys Phe Thr Cys Ile Ile Cys Asn 435 440 445 Asn LysLeu Arg Gly Gln Pro Phe Tyr Ala Val Glu Lys Lys Ala Tyr 450 455 460 CysGlu Pro Cys Tyr Ile Asn Thr Leu Glu Gln Cys Asn Val Cys Ser 465 470 475480 Lys Pro Ile Met Glu Arg Ile Leu Arg Ala Thr Gly Lys Ala Tyr His 485490 495 Pro His Cys Phe Thr Cys Val Met Cys His Arg Ser Leu Asp Gly Ile500 505 510 Pro Phe Thr Val Asp Ala Gly Gly Leu Ile His Cys Ile Glu AspPhe 515 520 525 Glu Lys Lys Phe Ala Pro Arg Cys Ser Val Cys Lys Glu ProIle Met 530 535 540 Pro Ala Pro Gly Gln Glu Glu Thr Val Arg Ile Val AlaLeu Asp Arg 545 550 555 560 Asp Phe His Val His Cys Tyr Arg Cys Glu AspCys Gly Gly Leu Leu 565 570 575 Ser Glu Gly Asp Asn Gln Gly Cys Tyr ProLeu Asp Gly His Ile Leu 580 585 590 Cys Lys Thr Cys Asn Ser Ala Arg IleArg Val Leu Thr Ala Lys Ala 595 600 605 Ser Thr Asp Leu 610 4067 basepairs nucleic acid single linear cDNA 164 CTTGAATCTT GGGGCAGGAACTCAGAAAAC TTCCAGCCCG GGCAGCCCGC GTTTGGTGCA 60 AGACTCAGGA GCTAGCAGCCCGTCCCCCTC CGACTCTCCG GTGCCGTTGC TGCCTGCTCC 120 CGCCACCCTA GGAGGCGCGGTGCCACCCAC TACTCTGTCC TCTGCCTGTG CTCCGTGCCC 180 GACCCTATCC CGGCGGAGTCTCCCCATCCT CCTTTGCTTT CCGACTGCCC AAGGCACTTT 240 CAATCTCAAT CTCTTCTCTCTCTCTCTCTC TCTCTCTGTC TCTCTCTCTC TCTCTCTCTC 300 TCTCTCTCTC GCAGGGTGGGGGGAAGAGGA GGAGGAATTC TTTCCCCGCC TAACATTTCA 360 AGGGACCACA ATCACTCCAAGTCTCTTCCC TTTCCAAGCC GCTTCCGAAG TGCTCCCGGT 420 GCCCGCAACT CCTGATCCCAACCCGCGAGA GGAGCCTCTG CGACCTCAAA GCCTCTCTTC 480 CTTCTCCCTC GCTTCCCTCCTCCTCTTGCT ACCTCCACCT CCACCGCCAC CTCCACCTCC 540 GGCACCCACC CACCGCCGCCGCCGCCACCG GCAGCGCCTC CTCCTCTCCT CCTCCTCCTC 600 CCCTCTTCTC TTTTTGGCAGCCGCTGGACG TCCGGTGTTG ATGGTGGCAG CGGCGGCAGC 660 CTAAGCAACA GCAGCCCTCGCAGCCCGCCA GCTCGCGCTC GCCCCGCCGG CGTCCCCAGC 720 CCTATCACCT CATCTCCCGAAAGGTGCTGG GCAGCTCCGG GGCGGTCGAG GCGAAGCGGC 780 TGCAGCGGCG GTAGCGGCGGCGGGAGGCAG GATGAGCGCA CGCGGTGAGG GCGCGGGGCA 840 GCCGTCCACT TCAGCCCAGGGACAACCTGC CGCCCCAGCG CCTCAGAAGA GAGGACGCGG 900 CCGCCCCAGG AAGCAGCAGCAAGAACCAAC CGGTGAGCCC TCTCCTAAGA GACCCAGGGG 960 AAGACCCAAA GGCAGCAAAAACAAGAGTCC CTCTAAAGCA GCTCAAAAGA AAGCAGAAGC 1020 CACTGGAGAA AAACGGCCAAGAGGCAGACC TAGGAAATGG CCACAACAAG TTGTTCAGAA 1080 GAAGCCTGCT CAGGAGGAAACTGAAGAGAC ATCCTCACAA GAGTCTGCCG AAGAGGACTA 1140 GGGGGCGCAA CGTTCGATTTCTACCTCAGC AGCAGTTGGA TCTTTTGAAG GGAGAAGACA 1200 CTGCAGTGAC CACTTATTCTGTATTGCCAT GGTCTTTCCA CTTTCATCTG GGGTGGGGTG 1260 GGGTGGGGTG GGGGAGGGGGGGGTGGGGTG GGGAGAAATC ACATAACCTT AAAAAGGACT 1320 ATATTAATCA CCTTCTTTGTAATCCCTTCA CAGTCCCAGG TTTAGTGAAA AACTGCTGTA 1380 AACACAGGGG ACACAGCTTAACAATGCAAC TTTTAATTAC TGTTTTCTTT TTTCTTAACC 1440 TACTAATAGT TTGTTGATCTGATAAGCAAG AGTGGGCGGG TGAGAAAAAC CGAATTGGGT 1500 TTAGTCAATC ACTGCACTGCATGCAAACAA GAAACGTGTC ACACTTGTGA CGTCGGGCAT 1560 TCATATAGGA AGAACGCGGTGTGTAACACT GTGTACACCT CAAATACCAC CCCAACCCAC 1620 TCCCTGTAGT GAATCCTCTGTTTAGAACAC CAAAGATAAG GACTAGATAC TACTTTCTCT 1680 TTTTCGTATA ATCTTGTAGACACTTACTTG ATGATTTTTA ACTTTTTATT TCTAAATGAG 1740 ACGAAATGCT GATGTATCCTTTCATTCAGC TAACAAACTA GAAAAGGTTA TGTTCATTTT 1800 TCAAAAAGGG AAGTAAGCAAACAAATATTG CCAACTCTTC TATTTATGGA TATCACACAT 1860 ATCAGCAGGA GTAATAAATTTACTCACAGC ACTTGTTTTC AGGACAACAC TTCATTTTCA 1920 GGAAATCTAC TTCCTACAGAGCCAAAATGC CATTTAGCAA TAAATAACAC TTGTCAGCCT 1980 CAGAGCATTT AAGGAAACTAGACAAGTAAA ATTATCCTCT TTGTAATTTA ATGAAAAGGT 2040 ACAACAGAAT AATGCATGATGAACTCACCT AATTATGAGG TGGGAGGAGC GAAATCTAAA 2100 TTTCTTTTGC TATAGTTATACATCAATTTA AAAAGCAAAA AAAAAAAGGG GGGGGCAATC 2160 TCTCTCTGTG TCTTTCTCTCTCTCTCTCCC TCTCCCTCTC TCTTTTCATG TGTATCAGTT 2220 TCCATGAAAG ACCTGAATACCACTTACCTC AAATTAAGCA TATGTGTTAC TTCAAGTAAT 2280 ACGTTTTGAC ATAAGATGGTTGACCAAGGT GCTTTTCTTC GGCTTGAGTT CACCATCTCT 2340 TCATTCAAAC TGCACTTTTAGCCAGAGATG CAATATATCC CCACTACTCA ATACTACCTC 2400 TGAATGTTAC AACGAATTTACAGTCTAGTA CTTATTACAT GCTGCTATAC ACAAGCAATG 2460 CAAGAAAAAA ACTTACTGGGTAGGTGATTC TAATCATCTG CAGTTCTTTT TGTACACTTA 2520 ATTACAGTTA AAGAAGCAATCTCCTTACTG TGTTTCAGCA TGACTATGTA TTTTTCTATG 2580 TTTTTTTAAT TAAAAATTTTTAAAATACTT GTTTCAGCTT CTCTGCTAGA TTTCTACATT 2640 AACTTGAAAA TTTTTTAACCAAGTCGCTCC TAGGTTCTTA AGGATAATTT TCCTCAATCA 2700 CACTACACAT CACACAAGATTTGACTGTAA TATTTAAATA TTACCCTCCA AGTCTGTACC 2760 TCAAATGAAT TCTTTAAGGAGATGGACTAA TTGACTTGCA AAGACCTACC TCCAGACTTC 2820 AAAAGGAATG AACTTGTTACTTGCAGCATT CATTTGTTTT TTCAATGTTT GAAATAGTTC 2880 AAACTGCAGC TAACCCTAGTCAAAACTATT TTTGTAAAAG ACATTTGATA GAAAGGAACA 2940 CGTTTTTACA TACTTTTGCAAAATAAGTAA ATAATAAATA AAATAAAGCC AACCTTCAAA 3000 GAACTTGAAG CTTTGTAGGTGAGATGCAAC AAGCCCTGCT TTTGCATAAT GCAATCAAAA 3060 ATATGTGTTT TTAAGATTAGTTGAATATAA GAAAATGCTT GACAAATATT TTCATGTATT 3120 TTACACAAAT GTGATTTTTGTAATATGTCT CAACCAGATT TATTTTAAAC GCTTCTTATG 3180 TAGAGTTTTT ATGCCTTTCTCTCCTAGTGA GTGTGCTGAC TTTTTAACAT GGTATTATCA 3240 ACTGGGCCAG GAGGTAGTTTCTCATGACGG CTTTTGTCAG TATGGCTTTT AGTACTGAAG 3300 CCAAATGAAA CTCAAAACCATCTCTCTTCC AGCTGCTTCA GGGAGGTAGT TTCAAAGGCC 3360 ACATACCTCT CTGAGACTGGCAGATCGCTC ACTGTTGTGA ATCACCAAAG GAGCTATGGA 3420 GAGAATTAAA ACTCAACATTACTGTTAACT GTGCGTTAAA TAAGCAAATA AACAGTGGCT 3480 CATAAAAATA AAAGTCGCATTCCATATCTT TGGATGGGCC TTTTAGAAAC CTCATTGGCC 3540 AGCTCATAAA ATGGAAGCAATTGCTCATGT TGGCCAAACA TGGTGCACCG AGTGATTTCC 3600 ATCTCTGGTA AAGTTACACTTTTATTTCCT GTATGTTGTA CAATCAAAAC ACACTACTAC 3660 CTCTTAAGTC CCAGTATACCTCATTTTTCA TACTGAAAAA AAAAGCTTGT GGCCAATGGA 3720 ACAGTAAGAA CATCATAAAATTTTTATATA TATAGTTTAT TTTTGTGGGA GATAAATTTT 3780 ATAGGACTGT TCTTTGCTGTTGTTGGTCGC AGCTACATAA GACTGGACAT TTAACTTTTC 3840 TACCATTTCT GCAAGTTAGGTATGTTTGCA GGAGAAAAGT ATCAAGACGT TTAACTGCAG 3900 TTGACTTTCT CCCTGTTCCTTTGAGTGTCT TCTAACTTTA TTCTTTGTTC TTTATGTAGA 3960 ATTGCTGTCT ATGATTGTACTTTGAATCGC TTGCTTGTTG AAAATATTTC TCTAGTGTAT 4020 TATCACTGTC TGTTCTGCACAATAAACATA ACAGCCTCTG TGATCCC 4067

1. Multi-tumor Aberrant Growth (MAG) gene having the nucleotide sequenceof any one of the strands of any one of the members of the High MobilityGroup protein genes or LIM protein genes, including modified versionsthereof.
 2. Multi-tumor Aberrant Growth (MAG) gene as claimed in claim 1having essentially the nucleotide sequence of the HMGI-C gene asdepicted in FIG. 7, or the complementary strand thereof, includingmodified or elongated versions of both strands.
 3. Multi-tumor AberrantGrowth (MAG) gene as claimed in claim 1 having essentially thenucleotide sequence of the LPP gene as depicted in FIG. 5, or thecomplementary strand thereof, including modified or elongated versionsof both strands.
 4. Multi-tumor Aberrant Growth (MAG) gene as claimed inclaim 1, 2 or 3 for use as a starting point for designing suitableexpression-modulating compounds or techniques for the treatment ofnon-physiological proliferation phenomena in human or animal. 5.Multi-tumor Aberrant Growth (MAG) gene as claimed in claim 1, 2 or 3 foruse as a starting point for designing suitable nucleotide probes for(clinically/medically) diagnosing cells having a non-physiologicalproliferative capacity as compared to wildtype cells.
 6. Protein encodedby the Multi-tumor Aberrant Growth (MAG) gene as claimed in claim 1, 2or 3 for use as a starting point for preparing suitable antibodies for(clinically/medically) diagnosing cells having a non-physiologicalproliferative capacity as compared to wildtype cells.
 7. Derivatives ofthe MAG gene as claimed in claim 1, 2 or 3 or of its immediate vicinityfor use in diagnosis and the preparation of therapeutical compositions,wherein the derivatives are selected from the group consisting of senseand anti-sense cDNA or fragments thereof, transcripts of the gene orpractically usable fragments thereof, antisense RNA, fragments of thegene or its complementary strand, proteins encoded by the gene orfragments thereof, antibodies directed to the gene, the cDNA, thetranscript, the protein or the fragments thereof, as well as antibodyfragments.
 8. In situ diagnostic method for diagnosing cells having anon-physiological proliferative capacity, comprising at least some ofthe following steps: a) designing a set of nucleotide probes based onthe information obtainable from the nucleotide sequence of the MAG geneas claimed in claim 1 or 2, wherein one of the probes is hybridisable toa region of the aberrant gene substantially mapping at the same locus asa corresponding region of the wildtype gene and the other probe ishybridisable to a region of the aberrant gene mapping at a differentlocus than a corresponding region of the wildtype gene; b) incubatingone or more interphase or metaphase chromosomes or cells having anon-physiological proliferative capacity, with the probe underhybridising conditions; and c) visualising the hybridisation between theprobe and the gene.
 9. Method of diagnosing cells having anon-physiological proliferative capacity, comprising at least some ofthe following steps: a) taking a biopsy of the cells to be diagnosed; b)isolating a suitable MAG gene-related macromolecule therefrom; c)analysing the macromolecule thus obtained by comparison with a wildtypereference molecule preferably from the same individual.
 10. Method asclaimed in claim 9, comprising at least some of the following steps: a)taking a biopsy of the cells to be diagnosed; b) extracting total RNAthereof; c) preparing at least one first strand cDNA of the mRNA speciesin the total RNA extract, which cDNA comprises a suitable tail; d)performing a PCR and/or RT-PCR using a MAG gene specific primer and atail-specific and/or partner-specific/nested primer in order to amplifyMAG gene specific cDNA's; e) separating the PCR products on a gel toobtain a pattern of bands; f) evaluating the presence of aberrant bandsby comparison to wildtype bands, preferably originating from the sameindividual.
 11. Method as claimed in claim 9, comprising at least someof the following steps: a) taking a biopsy of the cells to be diagnosed;b) isolating total protein therefrom; c) separating the total protein ona gel to obtain essentially individual bands and optionally trnasferringthe bands to a Western blot; d) hybridising the bands thus obtained withantibodies directed against a part of the protein encoded by theremaining part of the MAG gene and against a part of the protein encodedby the substitution part of the MAG gene; e) visualising theantigen-antibody reactions and establishing the presence of aberrantbands by comparison with bands from wildtype proteins, preferablyoriginating from the same individual.
 12. Method as claimed in claim 9,comprising at least some of the following steps: a) taking a biopsy ofthe cells to be diagnosed; b) isolating total DNA therefrom; c)digesting the DNA with one or more so-called “rare cutter” restrictionenzymes; d) separating the digest thus prepared on a gel to obtain aseparation pattern; e) optionally transfering the separation pattern toa Southern blot; f) hybridising the separation pattern in the gel or onthe blot with one or more informative probes under hybridisingconditions; g) visualising the hybridisations and establishing thepresence of aberrant bands by comparison to wildtype bands, preferablyoriginating from the same individual.
 13. Method as claimed in claim 9,comprising at least some of the following steps: a) taking a biopsy ofthe cells to be diagnosed; b) extracting mRNA therefrom; c) establishingthe presence or the (relative) quantity of mRNA derived from the MAGgene; and d) comparing the result of step c) with the result of asimilar experiment with wildtype cells, preferably originating from thesame individual.
 14. Method as claimed in any one of the claims 8-13,wherein the cells having a non-physiological proliferative capacity areselected from the group consisting of the mesenchymal tumors hamartomas(e.g. breast and lung), adipose tissue tumors (e.g. lipomas),pleomorphic salivary gland adenomas, uterine leiomyomas, angiomyxomas,fibroadenomas of the breast, polyps of the endometrium, atheroscleroticplaques, and other benign tumors as well as various malignant tumors,including but not limited to sarcomas (e.g. rhabdomyosarcoma,osteosarcoma) and carcinomas (e.g. of breast, lung, skin, thyroid), andhaematological malignancies, like leukemias and lymphomas. 15.Anti-sense molecules of a MAG gene as claimed in claim 1, 2 or 3 for usein the treatment of diseases involving cells having a non-physiologicalproliferative capacity by modulating the expression of the gene. 16.Expression modulators, such as inhibitors or enhancers, includingribozymes, of the MAG gene as claimed in claim 1, 2 or 3 for use in thetreatment of diseases involving cells having a non-physiologicalproliferative capacity.
 17. Antisense RNA molecules complementary to themRNA molecules of the MAG gene and/or antibodies directed against thegene product of the MAG gene as claimed in claim 1, 2 or 3 for use inthe treatment of diseases involving cells having a non-physiologicalproliferative capacity.
 18. Diagnostic kit for performing the method asclaimed in claim 8, comprising a suitable set of labeled nucleotideprobes.
 19. Diagnostic kit for performing the method as claimed in claim10, comprising a suitable set of labeled probes.
 20. Diagnostic kit forperforming the method as claimed in claim 11, comprising a suitable setof labeled MAG gene specific and tail specific PCR primers. 21.Diagnostic kit for performing the method as claimed in claim 11,comprising a suitable set of labeled probes, and suitable rare cuttingrestriction enzymes.
 22. Pharmaceutical composition for lowering theexpression level of the MAG gene in cells having a non-physiologicalproliferative capacity, comprising one or more of the derivatives asclaimed in claim 7 and/or one or more expression modulators as claimedin claim
 16. 23. Pharmaceutical composition as claimed in claim 22,wherein the cells having a non-physiological proliferative capacity areselected from the group consisting of the mesenchymal tumors hamartomas(e.g. breast and lung), adipose tissue tumors (e.g. lipomas),pleomorphic salivary gland adenomas, uterine leiomyomas, angiomyxomas,fibroadenomas of the breast, polyps of the endometrium, atheroscleroticplaques, and other benign tumors as well as various malignant tumors,including but not limited to sarcomas (e.g. rhabdomyosarcoma,osteosarcoma) and carcinomas (e.g. of breast, lung, skin, thyroid), andhaematological malignancies, like leukemias and lymphomas.
 24. Use ofthe derivatives as claimed in claim 7 for the preparation of adiagnostic kit or a pharmaceutical composition for the diagnosis ortreatment of diseases or disorders involving cells having anon-physiological proliferative capacity.
 25. Use of the expressionmodulators as claimed in claim 16 for the preparation of apharmaceutical composition for the treatment of diseases or disordersinvolving cells having a non-physiological proliferative capacity. 26.Use as claimed in claim 24 or 25, wherein the cells having anon-physiological proliferative capacity are selected from the groupconsisting of the mesenchymal tumors hamartomas (e.g. breast and lung),adipose tissue tumors (e.g. lipomas), pleomorphic salivary glandadenomas, uterine leiomyomas, angiomyxomas, fibroadenomas of the breast,polyps of the endometrium, atherosclerotic plaques, and other benigntumors as well as various malignant tumors, including but not limited tosarcomas (e.g. rhabdomyosarcoma, osteosarcoma) and carcinomas (e.g. ofbreast, lung, skin, thyroid), and haematological malignancies, likeleukemias and lymphomas.
 27. Method for isolating other MAG genes basedon the existence of a fusion gene, fusion transcript or fusion proteinin a tumor cell by using at least a part of a MAG gene for designingmolecular tools (probes, primers etc.).
 28. MAG genes obtainable by themethod of claim
 27. 29. MAG genes as claimed in claim 28 for use indiagnostic or therapeutic methods.
 30. Animal model for the assessmentof the utility of compounds or compositions in the treatment of diseasesor disorders involving cells having a non-physiological proliferativecapacity, which animal is a transgenic animal harbouring a MAG gene inits genome.
 31. Animal model as claimed in claim 30, wherein the MAGgene is an aberrant MAG gene, such as a fusion product of the remainingpart of the gene and the substitution part of its translocation partner.32. Animal model as claimed in claim 30, wherein the MAG gene shows anon-physiological expression level.
 33. Animal model for the assessmentof the utility of compounds or compositions in the treatment of diseasesor disorders involving cells having a non-physiological proliferativecapacity, which animal harbours a specific genetic aberration affectinga MAG gene as claimed in claim 1, 2 or 3 in the genome of at least partof its cells, which aberration is induced via homologous recombinationin embryonic stem cells.
 34. Animal model as claimed in any one of theclaims 30-33, which animal is a mammal, in particular a mouse, rat, dog,pig or higher primate, like chimpanzee.
 35. Poly- or oligonucleotideprobes and primers as disclosed in the description and figures.